Progress 08/21/12 to 08/20/17
Outputs
Target Audience:The target audience is represented by a broad research community of biologists working in the fields of neurobiology, muscle and heart biology, genetics, glycobiology & biochemistry, molecular & cellular biology, and biophysics. Our research data are expected to be also valuable for researchers working in fields of Biomedical Sciences, including areas of genetics and physiology of neurological diseases and muscular dystrophies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for training 5 undergraduate student researchers, 3 PhD students, and 2 postdoctoral trainees. Job opportunities have been provided for 3 student workers. How have the results been disseminated to communities of interest?The results have been disseminated via research publications and presentations at research conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue research according to our previous plans.
Impacts
What was accomplished under these goals?
During the reporting period, we focused on the following specific activities within the frame of the major goals:1. Cellular and molecular mechanisms underlying the neural function of sialylation. We focused on the role of sialylation in the control of sodium voltage-gated channels. This role was analyzed using genetic in vivo approaches, which included generation of special strains with GFP-labeled Na+ channel. We were also elucidating the roles of sialylation genes in cellular stress responses and neurodegeneration. 2. Regulation of biochemical and genetic pathways of sialylation. We initiated experiments to investigate the sialylation pathway and its role in glia-neuron coupling. To this end, we employed genetic, cell-biological and physiological approaches. We also started experiments using a combination of biochemical and genetic strategies to identify and characterization sialylated glycoproteins. 3. Role of protein O-mannosylation in neural regulation. We started applying genetic and neurobiological methods to elucidate involvement of POMT1/2 in targeting axonal connections, the control of sensory circuits and neuromuscular functions. 4. To unveil molecular targets of protein O-mannosylation. Experiments using a combination of biochemical and genetic approaches are underway to characterize functional targets of POMT1/2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Protein O-mannosyltransferases affect sensory axon wiring and dynamic chirality of body posture in the Drosophila embryo. Baker R, Nakamura N, Chandel I, Howell B, Lyalin D, Panin VM. J Neurosci. 2017 Nov 22. pii: 0346-17. doi: 10.1523/JNEUROSCI.0346-17.2017. [Epub ahead of print] PMID: 29167399
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Our target audience is a broad research community of biologists working in the fields of neurobiology, muscle and heart biology, genetics, glycobiology and biochemistry, molecular & cellular biology, and biophysics. Our research data are expected to be also valuable for researchers working in several fields of Biomedical Sciences, including areas of genetics and physiology of neurological diseases and muscular dystrophies. Changes/Problems:No major changes or modifications to our original research plans were made during reporting period. We have been successfully solving experimental problems that we encountered by employing alternative experimental strategies. What opportunities for training and professional development has the project provided?The project provided training and job opportunities for 2 postdoctoral trainees, 4 graduate student, and 7 undergraduate students. Participants: Postdoctoral trainees: Dr. Ilya Mertsalov (completed his training in February 2015), Dr. Boris Novikov. PhD Students: Hilary Scott (started her training in July 2011), Ryan Baker (started in January 2013), Brooke Howell (started in May 2013), Ishita Chandel (started in January 2015). Undergraduate student researchers: Jorge Galvan (Biochemistry Major), Zachary Cozzi (Biochemistry major), Nada Radwan (Nutrition and Food Science major), Maya Hogea (Biology major), Laura Haslam (Biochemistry major), Michael Jalfon (Biochemistry major), Maria Lyuksyutova (Biology major). Undergraduate student workers: Megan Hall (Animal Sciences major), Jeannette Flores (Geosciences major) and Zacchary Cozzi (Biochemistry major), Michael Jalfon (Biochemistry major), Kacy Petersen (Genetics major), Bhavana Kota (Biology major). How have the results been disseminated to communities of interest?Our results were disseminated via publications, conference presentations, invited seminar visits to other universities, and collaborative research. Publications during reporting period Scott H., Novikov B., Mertsalov I., Caster C., and Panin V. (2015) Novel regulatory mechanisms controlling sialylation in Drosophila nervous system. Glycobiology 25: 1298. Panin V., Baker R, Nakamura N., Leon-Aparicio D., and Guerrero-Hernandez A. Pathogenic mechanisms associated with abnormal protein O-mannosylation: Cell culture and Drosophila models. Glycobiology 25(11): 1308. Invited Seminars "Pathogenic mechanisms of protein O-mannosylation defects", March 2015. Children's National Medical Center, Washington, DC. Participation in research conferences: 2015 Annual Meeting of the Society for Glycobiology, San Francisco, December 2015. Consortium for Functional Glycomics Annual Satellite Meeting, San Francisco, December 2015. Collaborations included research projects that were performed in cooperation with researchers at Texas A&M (Dr. Zoran, Biology Department; Dr. Vladislav Yakovlev, Biomedical Engineering), and with scientists form other universities in the US and abroad (Dr. Wells and Dr. Tiemeyer, University of Georgia, Athens; Dr, Krylov, York University, Toronto, Canada; Dr. Richard Benton, UNIL, Switzerland; Dr. Guerrero-Hernandez, CINVESTAV-IPN, Mexico). What do you plan to do during the next reporting period to accomplish the goals?We will continue our project essentially according to our original plans, including the following research goals: (1) To characterize the function of sialylation using genetic, molecular, and physiological approaches. (2) To identify and characterize the in vivo targets of sialylation (3) To investigate the function of sialylation in the regulation of the blood-brain barrier and neural plasticity. (4) To reveal the regulation of sialylation and its interplay with galectin functions. (5) To elucidate the role of O-mannosylation in the regulation of neuromuscular development and control of muscle contractions by the peripheral nervous system.
Impacts
What was accomplished under these goals?
In the past year, we focused on the following Specific Activates: Aim 1: We focused on in vivo approaches to reveal how sialylation genes are regulated. We continued the detailed analyses of phenotypes of sialylation mutants. Aim 2: We focused on functional analyses of CSAS using in vitro and in vivo assays. We tested the hypothesis that CSAS and DSiaT may function as a molecular complex. We continued our experiments with Para using genetic approaches. Aim 3: We analyzed the plasticity of larval NMJs in DSiaT mutants using a paradigm of activity-dependent plasticity. Aim 4: A panel of genetic interaction assays was applied to elucidate the functional relationship between Galectin and sialylation genes. We also performed a site-specific recombinase based mutagenesis to generate mutations in galectin genes. Aim 5: In vivo analyses of mutants with Dystroglycan glycosylation defects have been carried out. The following significant results were obtained during the reporting period within the frame of our research goals: Aim 1: Our detailed characterization of sialylation mutants unveiled novel phenotypes indicating that sialylation is required for (i) maintenance of the blood-brain barrier, (ii) normal cardiac functions, including heart rate and rhythmic heart contraction, (iii) proper circadian behavior, including periods of resting and sleeping behaviors. With the goal to investigate how sialylation is regulated in the CNS during development, we created a transgenic strain with BAC construct containing a large genomic region encompassing the CSAS locus and encoding a FLAG-tagged version of CSAS. The functionality of CSAS BAC was confirmed in vivo by rescue experiments. This construct will be valuable for the analyses of CSAS localization within the CNS. Aim 2: We focused on functional analyses of CSAS using in vitro approaches. To this end, we developed a system for CSAS purification using a FLAG-tagged construct expressed in vivo or in Drosophila tissue culture. We purified CSAS with FLAG-affinity beads and started to characterize its enzymatic activity. To test the catalytic mechanism, we created and analyzed the CSAS-DA mutant with a single-site D(226)A substitution at a residue that was hypothesized to be a part of the CSAS catalytic site. We found that the mutant is expressed well but not active in vivo, which supported the hypothesis that D226 plays a key role in the CSAS catalytic reaction. The results of co-immunoprecipitation experiments did not support the hypothesis of molecular interaction between CSAS and DSiaT, which prompted further investigation of other possible scenarios of collaboration between CSAS and DSiaT. Additionally, we continued experiments with Para channel aimed at the analysis of its glycosylation. We have built transgenic strains for Para expression in genotypes with blocked or upregulated sialylation. Aim 3: We analyzed the plasticity of larval NMJs using a population density paradigm (plasticity associated with increased NMJ activity). We found that DSiaT mutants have a defect in NMJ plasticity as revealed by suppressed ability to increase the number of synaptic boutons and branches (Fig. 2). We are currently elucidating the molecular and genetic players that collaborate with sialylation in the control of NMJ plasticity. Aim 4: Our results indicated that mutations in galectin genes can suppress the paralysis phenotype of sialylation mutants. These data suggested that sialic acids function as a masking modifications that can regulate interactions between terminal Gal residues of glycans and Galectins. We also generated several mutants with potential deletions of genomic region including two galectin genes. Further molecular analyses of the mutant stocks have been initiated. Aim 5: Our analyses of the connection between sialylation and other glycosylation pathways revealed the involvement of O-mannosylated proteins beside Dystroglycan in the regulation of neuromuscular functions. We also found that O-mannose modification pathway has both Dystroglycan-dependent and independent functions. Several novel putative targets of O-mannosylation have been identified, including Receptor Protein Tyrosine Phosphatases (RPTPs).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Scott H., Novikov B., Mertsalov I., Caster C., and Panin V. (2015) Novel regulatory mechanisms controlling sialylation in Drosophila nervous system. Glycobiology 25: 1298.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Panin V., Baker R, Nakamura N., Leon-Aparicio D., and Guerrero-Hernandez A. Pathogenic mechanisms associated with abnormal protein O-mannosylation: Cell culture and Drosophila models. Glycobiology 25(11): 1308.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The results we generated in our projects are valuable for a broad community of biologists working in the fields of neurobiology, muscle and heart biology, genetics, glycobiology and biochemistry, molecular & cellular biology. Moreover, our research data can be important for other researchers working in the area of Biomedical Sciences, including genetics and physiology of neurological diseases and muscular dystrophies. Changes/Problems: There were no major modifications during reporting period. The experimental problems that we encountered during the reporting period were solved, or alternative strategies were developed to overcome the obstacles (e.g., we have developed and are currently implementing a new strategy to generate galectin mutants). We continue carrying out the project essentially according to our original plans. What opportunities for training and professional development has the project provided? The project provided training and job opportunities for 2 postdoctoral trainees, 7 graduate student (including 3 rotation students), and 8 undergraduate students. How have the results been disseminated to communities of interest? Our results were included publications, conference presentations, and disseminated in invited seminar visits to other universities (including Department of Biochemistry, CINVESTAV, Mexico City, Mexico, and outreach and Department of Biological Sciences, Vanderbilt University, Nashville, TN) Our dissemination activities also included several collaboration that we actively pursued during the reporting period, including collaborations within Texas A&M (Dr. Zoran's laboratory, Biology Department) and with other universities in the US and abroad (Dr. Wells and Dr. Tiemeyer, University of Georgia, Athens; Dr, Krylov, York University, Toronto, Canada; Dr. Richard Benton, UNIL, Switzerland; Dr. Guerrero-Hernandez, CINVESTAV-IPN, Mexico). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the reporting period, we obtained the following results within the frame of our research goals: (1) To characterize the function of sialylation using genetic, molecular, and electrophysiological approaches. The focus of our investigation was on the regulation of the sialylation pathway. We used genetic and biochemical approaches to characterize the function of CMP-sialic acid synthetase (CSAS) gene. We analyzed the phenotypes of previously generated mutants and created several novel mutant and transgenic genotypes to shed light on the role of sialylation in neural regulation. We continued investigation of subcellular localization of CSAS using in vivo, cell culture, and in vitro approaches. We are currently preparing a manuscript based on these new results (Mertsalov et al. Functional characterization of Drosophila CSAS. In preparation). To identify and characterize the in vivo molecular targets of DSIAT. We continued applying genetic strategies for revealing functional partners and potential targets of sialylation genes. From the list of candidate genes revealed in our laboratory, as well as by the group of our collaborator, Dr. Tiemeyer at the University of Georgia (Athens, GA), we focused on neurexin. Our preliminary data indicate genetic interactions between neurexin and DSiaT. Further experiments are underway to characterize endogenously sialylated glycoproteins using a combination of genetic and biochemical approaches. (3) Function of sialylation in plasticity and neural cell adhesion. We continued investigation of role of sialylation in the blood-brain barrier (BBB) pathway. Our main efforts were focused on establishing a reliable and quantitative assay for measuring the integrity of the BBB. Our results confirmed that DSiaT mutations cause increased permeability of the BBB, which also suggested that this pathogenic mechanism could underlie the excitability defect that we previously characterized in sialylation mutants. We continued collaborative interactions with Dr. Benton (UNIL, Lausanne, Switzerland) aimed at elucidation of the role of sialylation in the olfactory system. (4) Interplay between galectin and sialylation pathways. We continued investigation of relations between the sialylation pathway and galectins. Using newly generated genetic stocks and re-designed strategy, we continue this line of experiments. (5) Dystroglycan as a target of glycosylation. We continued developing Drosophila model system for investigation of molecular and genetic mechanisms of muscular dystrophies associated with abnormal protein O-mannosylation. Our experiments revealed developmental and physiological pathomechanisms that underlie posture and muscle contraction defects the mutants with defective O-mannosylation. These results potentially shed light on new pathological mechanisms that are conserved in humans, and we currently preparing a publication with these data (Baker et al. Protein O-mannosylation is required for normal muscle contractions and the control of body posture in Drosophila. In preparation).
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Scott, H. and Panin, V.M. (2014) The role of protein N-glycosylation in neural transmission. Glycobiology, 24: 407-417
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Panin VM and Wells L (2014) Protein O-mannosylation in metazoan organisms. Curr Protoc Prot Sci 75: Unit 12.12.
- Type:
Book Chapters
Status:
Accepted
Year Published:
2014
Citation:
Scott, H. and Panin, V.M. (2014) N-glycosylation in the regulation of the nervous system neural transmission. Book chapter in Glycobiology of the Nervous System, R.K. Yu and C.-L. Schengrund, eds. Springer Science+Business Media, New York.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Scott H., Caster C., Mertsalov I., Alfert M., Howell B., Zoran M., and Panin V. (2014) The role of CMP-Sialic acid synthetase in Drosophila neural transmission. Glycobiology 24: 1159.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Baker R, Nakamura N., Lyalin D., Alfert M., Guerrero-Hernandez A., and Panin V. (2014) Developmental rules and pathogenic mechanisms associated with protein O-mannosylation in Drosophila. Glycobiology 24(11): 1190.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Our activities reached a broad community of biologists working in the fields of neurobiology, muscle and heart biology, genetics, glycobiology and biochemistry, molecular & cellular biology. In addition, our activities were valuable for researchers working in the field of Biomedical Sciences, including areas of genetics and physiology of neurological diseases and muscular dystrophies. Through participation in Physics and Engineering Festival, our lab reached local community, including many school children who attended the event. Changes/Problems: There were no major modifications during reporting period. We had some experimental problems (e.g., our initial experiments that attempted to generate mutations in two galectin genes were not successful). These problems were solved, and we carried out the project essentially according to our original plans. What opportunities for training and professional development has the project provided? The project provided training and job opportunities for 1 postdoctoral trainee, 5 graduate student, and 8 undergraduate students, including: Postdoctoral trainees: Dr. Ilya Mertsalov (started in February 2013). PhD Students: Hilary Scott (started her training in July 2011), Michelle Alfert (started in September 2013), Ryan Baker (started in January 2013), Brooke Howell (started in May 2013), and Courtney Caster (May –September 2013, as graduate student, January – May, as a Research Assistant). Undergraduate student researchers: Anuj Manandhar (Biochemistry/Genetics Major, graduated in May 2013 with undergraduate thesis from Honors Undergraduate Program), Thomas Hunt (Biochemistry major), Morgan Ritz (Biochemistry/Genetics major, graduated in May 2013 with undergraduate thesis), Haley Quinlan (Biochemistry/ Genetics major), Jennifer Suarez (Biochemistry major). The project also involved a Research Assistant (Courtney Caster). Undergraduate student workers: Nada Radwan (Nutrition major), Megan Hall (Animal Sciences major), and Zacchary Cozzi (Biochemistry major). How have the results been disseminated to communities of interest? Disseminationof the results included a publication in the Journal of Neuroscience (Islam et al, 2013). Additionally, our outreach activities included several collaboration that we actively pursued during the reporting period, including collaborations within Texas A&M (Dr. Zoran’s laboratory, Biology Department) and with other universities in the US and abroad (Dr. Wells and Dr. Tiemeyer, University of Georgia, Athens; Dr, Krylov, York University, Toronto, Canada; Dr. Richard Benton, UNIL, Switzerland; Dr. Guerrero-Hernandez, CINVESTAV-IPN, Mexico). What do you plan to do during the next reporting period to accomplish the goals? We will continue our project essentially as we planned, while focusing on the following research goals: (1) To characterize the function of sialylation using genetic, molecular, and physiological approaches. (2) To identify and characterize the in vivo targets of sialylation (3) To investigate the function of sialylation in neural cell adhesion and plasticity. (4) To reveal the regulation of sialylation and its interplay with galectin functions. (5) To elucidate the role of O-mannosylation in the function of dystroglycan and other molecular targets in muscles and the nervous system.
Impacts
What was accomplished under these goals?
We use Drosophila as a versatile and powerful model system to elucidate evolutionarily conserved mechanisms of glycosylation and their roles in cellular excitability, cell adhesion, and neural and muscular development and physiology. We use a multidisciplinary research strategy relying on approaches from different areas of modern biology, including biochemistry, developmental neurobiology, glycobiology, cell biology and molecular genetics. Our research sheds light on the mechanisms of sialylation in the nervous system of animals and evolutionarily conserved mechanisms of O-mannosylation that can prevent muscular dystrophy in humans. We investigate the regulation of these two glycosylation pathways and the functions of their main players using in vivo and in vitro approaches. Our results suggest that similar molecular and physiological mechanisms are involved in the regulation of analogous processes in humans, including neural transmission and muscular development and homeostasis. We presented and discussed our research in our peer-reviewed publication. The results were also disseminated via interactions with collaborators and peers working in related research fields. Our research has been supported by RO1 NIH grant. Additionally our experiments were supported by a CONACYT-TAMU grant that supported our collaborative project with Dr. Dr. Guerrero-Hernandez (CINVESTAV-IPN), and a Short Visits Research Grant from Swiss Science Foundation that supported our collaboration with Dr. Benton (UNIL, Switzerland). We share our protocols and other research information with a broader scientific community via publications, conference presentations, and personal communication. We make all reagents generated in our research (such as DNA constructs, engineered mutant and transgenic flies, tissue culture lines, etc.) freely available to other researchers for further exploration of the involvement of sialylation in neural development. One postdoctoral fellow, 5 graduate and 8 undergraduate students have been mentored, trained and supervised in the laboratory during reporting period. During the reporting period, we obtained the following results within the frame of our research goals: (1) To characterize the function of sialylation using genetic, molecular, and electrophysiological approaches. We investigated the regulation of neural excitability by DSiaT-mediated sialylation. Our focus was on the regulation of the sialylation pathway by the CMP-sialic acid synthetase (CSAS) gene. We analyzed the phenotypes of previously generated mutants and created several novel mutant and transgenic genotypes to shed light on the role of sialylation in neural regulation. We found that unlike mammalian homologs, the Drosophila CSAS protein is localized in the Golgi, which discovered a unique change in the regulation of the pathway at subcellular level during metazoan evolution. These new results were included in our publication (Islam et al. J Neurosci 2013). 2. To identify and characterize the in vivo molecular targets of DSIAT Using a panel of genetic interaction assays together with novel mutants generated in our laboratory and obtained from other laboratories, we identified several strongly interacting genetic partners of sialylation genes. They include voltage–gated channels, such as sodium channel Para and potassium channel Sei. In addition, in collaboration with the laboratory of Dr. Tiemeyer at the University of Georgia (Athens, GA), we obtained a list of candidate targets for sialylation in Drosophila. These proteins were included in the further genetic and biochemical experiments focused on elucidation of glycosylation of these proteins. (3) Function of sialylation in neural cell adhesion. We continued experiments focused on the role of sialylation in the maintenance of the blood-brain barrier (BBB). Our results suggested that DSiaT and CSAS can have separate functions in the BBB pathway since CSAS mutants do not have a significant defect in BBB permeability, while DSiaT mutations cause increased permeability of the BBB. Additionally, we started investigation the connection between the BBB and circadian rhythm regulation. Our preliminary results indicated that DSiaT mutants are significantly arrhythmic, suggesting a novel role for sialylation in the regulation of the circadian clock pathway. Additionally, in collaboration with Dr. Benton (UNIL, Lausanne, Switzerland) we obtained evidence that axonal connections of projection neurons of olfactory system show targeting defects in sialylation mutants, which indicated a potential role of sialylation in modulating neural cell adhesion molecules involved in axonal guidance. (4) Interplay between galectin and sialylation pathways. Our initial experiments that attempted to generate mutations in two galectin genes were not successful. We obtained new genetic stocks and re-designed our experimental strategy, which is expected to facilitate the generation of galectin mutants and accelerate the project which is currently in progress. (5) Dystroglycan as a target of glycosylation. We developed a new physiological model system based on live heart assays in flies. Using this system, we discovered heart contraction phenotypes in O-mannosylation mutants. We continue experiment to reveal developmental and physiological pathomechanisms that underlie the heart defects in the mutants. These results potentially shed light on new pathological mechanisms that are conserved in humans and can affect heart physiology in muscular dystrophy patients.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Islam R, Nakamura M, Scott H, Repnikova E, Carnahan M, Pandey D, Caster C, Khan S, Zimmermann T, Zoran MJ, Panin VM (2013) The role of Drosophila cytidine monophosphate-sialic acid synthetase in the nervous system. J Neurosci 33:12306-12315.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: During last year, our project was concentrated on the following aims: (1) To characterize the function of sialylation using genetic, molecular, and electrophysiological approaches. We previously concentrated on the last enzyme in the sialylation pathway, the Drosophila sialyltransferase gene. More recently, our project extended investigation to include other genes of this pathway. We have focused on CMP-Sialic acid synthetase that mediates penultimate step in the biosynthesis of sialic acids. We generated and characterized several mutant alleles of this gene. We also investigated the expression of this gene protein product, CSAS. We found that the CSAS and sialyltransferase genes have complex and unusual genetic interactions, suggesting novel mechanisms involved in the control of neural-specific sialylation. Our experiments have revealed a new regulation of neural excitability by glycosylation. (2) To identify and characterize the in vivo molecular targets of DSIAT. Our experiments were focused on voltage-gated sodium channel as putative molecule regulated by direct sialylation of its N-linked glycans. We generated transgenic Drosophila strains and tissue culture lines for expression and purification of this protein. Using a biochemical strategy, we currently investigate the structure of carbohydrate modifications of the channel protein in collaboration with Dr. Tiemeyer and Dr. Wells, experts in mass spectrometry approaches applied to glycoproteins and glycans. Initial data from these experiments have been presented at the Annual Glycobiology Conference in San Diego last fall. (3) Function of sialylation in neural cell adhesion. We continued experiments aimed at revealing the effect of sialylation on the function of neural cell adhesion. We specifically focused on neurexin and obtained preliminary data that its function in the maintenance of blood-brain barrier may be compromised in sialylation mutants. We are working on confirming this important result using genetic, physiological and pharmacological approaches. (4) Interplay between galectin and sialylation pathways. We continued research in this new direction. We are generating galectin mutants. Our genetic experiments confirmed the interactions between sialylation and galactosylation pathways. These new results have been published in G3: Genes Genomes Genetics. (5) Dystroglycan as a target of glycosylation. Defects in O-mannosylation of Dystroglycan (Dg) result in muscular dystrophies termed dystroglycanopathies. We have been developing a Drosophila model of dystroglycanopathies to investigate pathological mechanisms of these diseases using advantages of Drosophila model system. Our experiments also suggested a possibility of intersection between O-mannosylation and sialylation pathways that impinges on Dystroglycan posttranslational modifications. Our more recent experiments revealed the temporal and spatial requirement of O-mannosylation for the development of muscle attachment sites. Dissemination of the results include publications, presentations at conferences and outreach activities, including collaborations within Texas A&M and with other universities in the US and abroad. PARTICIPANTS: The project provided training opportunities for 1 postdoctoral trainee, 1 graduate student, and 6 undergraduate student researchers, including: Postdoctoral trainees: Dr. Dmitry Lyalin (October 2011-November 2012). PhD Students: Hilary Witzenman (started her training in July 2011). Undergraduate students: Niraj Kc (Biochemistry/Genetics Major, graduated in May 2012 with undergraduate thesis from Honors Undergraduate Program), Thomas Hunt (Biochemistry major), Morgan Ritz (Biochemistry/Genetics major), Haley Quinlan (Biochemistry/ Genetics major), Jennifer Suarez (Biochemistry major), Anuj Manandhar (Biochemistry/Genetics major, graduating with undergraduate research thesis from Honors Program in 2013). The project also involved a Research Assistant (Courtney Caster). TARGET AUDIENCES: Our research results will be important for a broad community of biologists working in the fields of neurobiology, muscle and heart biology, genetics, glycobiology and biochemistry, molecular & cellular biology. In addition, our results are valuable for researchers working in the field of Biomedical Sciences, including areas of genetics and physiology of neurological diseases and muscular dystrophies. PROJECT MODIFICATIONS: There were no major project modifications during reporting period.
Impacts
We employ Drosophila as a versatile and powerful model system to elucidate evolutionarily conserved mechanisms of glycosylation and their roles in cellular excitability, cell adhesion, and neuromuscular development and physiology. We use a multidisciplinary research strategy relying on approaches from different areas of modern biology, including biochemistry, developmental neurobiology, glycobiology, cell biology and molecular genetics. Our research sheds light on the mechanisms of sialylation in the nervous system of animals. We investigate the regulation of sialylation and the functions of main players of sialylation pathway using in vivo and in vitro approaches. Our results suggest that similar molecular and neurophysiological mechanisms are involved in the regulation of neural transmission in human brain. We presented and discussed our research in our peer-reviewed publications, at National and International conferences and Society meetings, such as the Annual Conference of the Society for Glycobiology (San Diego, CA) and Annual GlycoSymposium on Emerging Paradigms in Glycobiology (Athens, GA). The results were also disseminated via interactions with collaborators and peers working in related research fields. Our research has been supported by RO1 NIH grant. Our research data provided a basis for initiating collaboration with Dr. Dr. Guerrero-Hernandez (CINVESTAV-IPN) supported by CONACYT-TAMU grant. We share our protocols and other research information with a broader scientific community via publications, conference presentations, and personal communication. We make all reagents generated in our research (such as DNA constructs, engineered mutant and transgenic flies, tissue culture lines, etc.) freely available to other researchers for further exploration of the involvement of sialylation in neural development. One postdoctoral fellow, one graduate and 6 undergraduate students have been mentored in the laboratory during reporting period.
Publications
- Witzenman H, Lyalin D., Caster C, Nakamura M, Kc N, Islam R, Katoh T, Zoran M., Tiemeyer M, and Panin V. (2012) Nervous system-specific sialylation in Drosophila. Glycobiology 22(11): 1525.
- Nakamura M, Pandey D, Panin VM (2012) Genetic interactions between Drosophila sialyltransferase and 1,4-N-acetylgalactosaminyltransferase-A genes indicate their involvement in the same pathway. G3: Genes Genomes Genetics, 2(6): 653-656
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: During last year, our project was concentrated on the following aims: (1) To characterize the function of sialylation using genetic, molecular, and electrophysiological approaches. We recently extended our project to investigate other genes potentially involved in sialylation pathway. We generated mutants in the CSAS gene encoding CMP-Sialic acid synthetase, the enzyme that produces sugar donor for DSiaT-mediated sialylation. We found that CSAS mutants phenocopy the mutations in DSiaT, which supports the hypothesis that CSAS and DSiaT collaborate within the same pathway. We anticipate that our experiments will reveal a new paradigm of neural regulation by glycosylation. (2) To identify and characterize the in vivo molecular targets of DSIAT. We continued our search for functionally important targets of sialylation. We implemented two complementary strategies, using a candidate approach along with unbiased biochemical screen for sialylated glycoproteins. In the candidate approach, we focused on Para, voltage-gated Na+ channel. We have generated tagged Para constructs for in vivo and cell culture expression and purification. The biochemical screen approach, carried out in collaboration with Dr. Teiemeyer (University of Georgia, Athens), has revealed several potential targets of sialylation expressed in the nervous system. (3) Function of sialylation in neural cell adhesion. Our preliminary results revealed that that axonal wiring of projection neurons could be regulated by sialylation. This result suggested that the function of some adhesion molecules may depend on sialylation. We continue this line of experiments and included several cell-adhesion molecules that could affect axonal wiring on the list of potentially targets of sialylation. (4) Interplay between galectin and sialylation pathways. We have initiated new experiments to investigate the role of galectins in neural transmission. We are currently testing the hypothesis that neural transmission is regulated by interplay between galectin and sialylation pathways. (5) Dystroglycan as a target of glycosylation. Abnormal glycosylation of Dystroglycan (Dg) causes severe muscular dystrophies called dystroglycanopathies. We are using Drosophila as a model system to investigate molecular and genetic mechanisms of dystroglycanopathies. Dg also represents a potential target of DSiaT activity. Dissemination of the results include publications, presentations at conferences (invited oral presentations at the Consortium for Functional Glycomics Meeting, CCRC, University of Georgia, Athens; International Glyco21 Meeting, Vienna, Austria; invited talks at the University of Wyoming, Laramie WY, and the Ege University, Izmir, Turkey; poster presentations at the Annual Meeting of The Society for Glycobiology, Seattle WA) and outreach activities, including collaborations within Texas A&M (Dr. Zoran's laboratory, Biology Department) and with other universities (Dr. Wells and Dr. Tiemeyer, University of Georgia, Athens; Dr, Krylov, York University, Toronto, Canada; Dr. Eric Bennett, University of South Florida, Tampa, FL). PARTICIPANTS: The project provided training opportunities for 1 postdoctoral trainee, 4 graduate students, and four undergraduate student workers and researchers, including: Postdoctoral trainees: Dr. Dmitry Lyalin (started his training in October 2011). PhD Students: Mindy Carnahan (graduated in May 2011), Dmitry Lyalin (graduated in August 2011), and Hilary Witzenman (started her training in July 2011) Undergraduate students: Niraj Kc (Biochemistry/Genetics Major, will graduate in May 2012 with undergraduate thesis from Honors Undergraduate Program), Saba Khan (Biochemistry/Genetics major, graduated in December 2011), Thomas Hunt (Biochemistry major), Morgan Ritz (Biochemistry/Genetics major). The project also involved a Research Assistant (Dheeraj Pandey). TARGET AUDIENCES: Our research results will be important for a broad community of biologists working in the fields of neurobiology, muscle and heart biology, genetics, glycobiology and biochemistry, molecular & cellular biology. In addition, our results will be valuable for Biomedical Sciences, including researchers working on genetics and physiology of neurological diseases and on pharmacological approaches to ameliorate neuronal excitability defects and heart abnormalities, and clinical researchers in muscular dystrophy field. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Using Drosophila as a versatile model system, we elucidate evolutionarily conserved mechanisms of glycosylation and their roles in cellular excitability, cell adhesion, and neuromuscular development. We employ a multidisciplinary strategy of research, including approaches from several areas of modern biology, such as biochemistry, developmental neurobiology, glycobiology, cell biology and molecular genetics. Our research sheds light on the mechanisms of sialylation in the nervous system of animals. We investigate the regulation of sialylation and the functions of main players of sialylation pathway using in vivo and in vitro approaches. Our results suggest that similar molecular and neurophysiological mechanisms are involved in the regulation of neural transmission in human brain. We presented and discussed our research at National and International conferences and Society meetings, such as the Annual Conference of the Society for Glycobiology (Seattle, WA) and International Glyco 21 Symposium (Vienna, Austria). The results were also disseminated via interactions with collaborators. We used our results to prepare and submit application for RO1 NIH grant. This application was funded and it will support our further research on sialylation. We share our protocols and other research information with a broader scientific community via publications, conference presentations, and personal communication. We make all reagents generated in our research (such as DNA constructs, engineered mutant and transgenic flies, tissue culture lines, etc.) freely available to other researchers for further exploration of the involvement of sialylation in neural development. One postdoctoral fellow, three graduate and 4 undergraduate students have been mentored in the laboratory during reporting period.
Publications
- Witzenman H, Pandey D, Repnikova R, Nakamura M, Islam R, Kc N, Caster C, and Panin V. 2011 Genetic and functional mechanisms of Drosophila sialylation. Glycobiology 21(11): 1517-18.
- Katoh T, Panin V, Tiemeyer M. 2011 Sialylated glycoproteins of Drosophila melanogaster. Glycobiology 21(11): 1512.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: During last year, our project was concentrated on the following aims: (1) To characterize the function of DSiaT using genetic, molecular, and electrophysiological approaches. We found that DSiaT affects the function of voltage-gated Na+ channel, membrane excitability, and the development of neuromuscular junctions. Our data provided the first evidence that sialylation has an important biological function in invertebrates. We revealed that a simple N-linked glycoprotein sialylation plays a specific and prominent role in modulating neural activity, which established a new mechanism of the involvement of glycosylation in the nervous system regulation. This novel, nervous system-specific function of N-linked sialylated glycans is potentially conserved between flies and humans. We published the results of these experiments. (2) To identify and characterize the in vivo molecular targets of DSIAT. Using genetic interaction assays we found that upregulation of sialylation pathway can elevate neural transmission. intriguing synergistic interactions between CSAS, a novel gene potentially involved the production of donor substrate for DSiaT, and DSiaT. We generated mutant allele for CSAS. We are currently using this allele to confirm the functional importance of sialylation for Para activity by genetic assays. In parallel, we have initiated molecular analysis of Para glycosylation using transgenic approaches. We are generating tagged Para constructs for in vivo expression and purification. A publication on Para-DSiaT interaction has been submitted for publication. (3) The role of sialylation in neural plasticity. Using a combination of genetic and physiological approaches, we found evidence that sialylation regulates plasticity within the CNS. We applied a developmental heat shock paradigm and revealed that DSiaT mutants have defects of temperature-induced plasticity of larval NMJs. (4) Function of sialylation in neural cell adhesion. Our initial analysis of single-cell mutant clones of projection neurons indicated that axonal wiring of these neurons is regulated by DSiaT activity. DSiaT expression is initiated in olfactory neurons before they execute neurite wiring. We found that DSiaT mutations result in wiring phenotype of olfactory neurons, characterized by redistributions of axonal connections between two different brain areas. (5) Dystroglycan (Dg) as a target of glycosylation. Abnormal glycosylation of Dg causes severe muscular dystrophies called dystroglycanopathies. We started exploring possibility of using Drosophila as a model to investigate molecular and genetic mechanisms of dystroglycanopathies. Dg another molecular target of DSiaT. Recent experiments that we carried out collaboration with Dr. Wells (University of Georgia, Athens) suggested that glycosylation of Dg can has an effect on its conformation, thus affecting Dg-ligand interactions. We discussed this mechanism in our recently published review on evolutionary aspects of Dg glycosylation. PARTICIPANTS: So far the project provided training opportunities for 2 postdoctoral trainees and 2 graduate students, and a number of undergraduate student workers and researchers, including: Postdoctoral trainees: Dr. Naosuke Nakamura (completed his training in March 2010), Dr. Rafique Islam (completed his training in September 2010) PhD Students: Dmitry Lyalin (currently in the lab); Mindy Carnahan (currently in the lab), Elena Repnikova (graduated in August 2009) Undergraduate students: Dheeraj Pandey (Biochemistry/Genetics Major, graduated in May 2010, currently a Research Assistant in the lab), Apoorva Ambavane (Biochemistry/Genetics major, graduated in May 2010 with undergraduate thesis from Honors Undergraduate Scholars Program), Myrthala Molar (Biochemistry major), Saba Khan (Biochemistry/Genetics major), Tina Zimmermann (ACS-IREU NSF student from Berlin, Germany). The project also involved a Research Assistant (Michiko Nakamura). TARGET AUDIENCES: The outcomes of our project will be important for a broad community of basic scientists working in the fields of neurobiology, heart biology, genetics, glycobiology and biochemistry, molecular & cellular biology. In addition, our results will be valuable for Medical Sciences, including researchers working on genetics and physiology of neurological diseases and on pharmacological approaches to treatment of neuronal excitability defects and heart abnormalities, and the field of muscular dystrophy research. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Using Drosophila as a versatile model system, we shed light evolutionarily conserved mechanisms that underlie the function of glycosylation in cellular excitability, cell adhesion, and neuromuscular development. Applying multidisciplinary strategy of research, we shed light on the functions of sialylation in the nervous system of animals. We continued exploring the regulation of sialylation pathway and elucidating its major players using in vivo and in vitro approaches. Our results suggested that similar processes are involved in the regulation of neural transmission in human brain. The results of our research were presented at National and International conferences and Society meetings, such as the Annual Conference of the Society for Glycobiology (Tampa, FL) and International SialoGlyco 2010 Meeting (Berlin, Germany). The results were also disseminated via interactions with collaborators (a collaborative paper was published). We share our protocols and other research information with a broader scientific community via publications, conference presentations, and personal communication. We make all reagents generated in our research (such as DNA constructs, engineered mutant and transgenic flies, tissue culture lines, etc.) freely available to other researchers for further exploration of the involvement of sialylation in neural development. Two postdoctoral trainees and two PhD student have been mentored during reporting period.
Publications
- Nakamura, N., Lyalin, D., and Panin, V.M (2010) Protein O-mannosylation in animal development and physiology: from human disorders to Drosophila phenotypes. Semin. Cell. Dev. Biol. 21(6):622-30 [PMID: 20362685, PMC2917527, NIHMS220225]
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: During last year, our project was concentrated on the following aims: 1. To characterize the structure-function relationship of DSIAT protein. In vivo analysis of a point mutant H406K have revealed that mutation of H406 residue results in biologically inactive protein. The analysis of H406K protein indicated that the mutation of His 406 specifically affects enzymatic activity, while having no effect on the level of expression or subcellular localization of sialyltransferase in vivo. Our results suggest that His 406 is an essential residue of DSiaT catalytic site. 2. To comprehensively characterize the expression pattern of the DSiaT gene during development, and to determine the cellular and subcellular distribution of DSIAT. We found that a striking loss of DSIAT expression in aged mutant flies' brains is not associated with the decrease in DSiaT mRNA transcript, which suggested that DSiaT protein expression is downregulated at a posttranscriptional level. This new finding established a new direction of our research focused on elucidating the molecular mechanism of post-transcriptional regulation of DSIAT. This mechanism may be evolutionarily conserved in humans, and thus our experiments may shed light on similar regulation of human DSIAT homologue, ST6GAL II, in the human brain. 3. To characterize the mutant phenotype of DSiaT. We continued the analysis of DSiaT mutant phenotypes, while focusing on the electrophysiological defects of neuronal transmission at larval NMJ and on genetic interactions with genes potentially involved in DSiaT pathway, known paralytic mutants, as well as novel genes predicted to affect sialylation through their involvement in the production of activated sugar donor, CMP-Sia. We continued to explore the functional relationship between DSiaT and para, the gene encoding a subunit of sodium voltage-gated channel. To reveal a specific effect of DSiaT on Para function, we used pharmacological assays involving the analysis of the effect of TTX (tetrodotoxin) on evoked excitatory junction potential (EJP). We also characterized the sensitivity of DSiaT mutants to DDT, a common insecticide that specifically affect Para activity. We found that DSiaT mutants are more resistant to both TTX and DDT in these assays. Our experiments suggested a possibility that Para is a direct molecular target of DSiaT activity. We are currently exploring this possibility using molecular approaches (see Aim 4). Aim 4. To identify and characterize the in vivo molecular targets of DSIAT. PARTICIPANTS: The project provided training opportunities for 3 postdoctoral trainees, 4 graduate students, and a number of undergraduate student workers and researchers, including: Postdoctoral trainees: Dr. Naosuke Nakamura (currently in the lab), Dr. Rafique Islam (started in November 2009) PhD Students: Dmitry Lyalin (currently in the lab); Mindy Altmyer (currently in the lab), Elena Repnikova (graduated in August 2009) MS Student: David Sullivan Undergraduate students: William Payne (Biology major), Dheeraj Pandey (Biochemistry/Genetics Major, graduated in December 2009 with undergraduate thesis from Undergraduate Scholars Program), Apoorva Ambavane (Biochemistry/Genetics major, graduating in May 2010 with undergraduate thesis from Honors Undergraduate Scholars Program), Myrthala Molar, (Biochemistry major) The project also involved a Research Assistant (Michiko Nakamura). TARGET AUDIENCES: The outcomes of our project will be important for a broad community of basic scientists working in the fields of Neurobiology, Genetics, Glycobiology and Molecular & Cellular Biology. In addition, our results will be valuable for Medical Sciences, including researchers working on genetics and physiology of neurological diseases and on pharmacological approaches to treatment of neuronal excitability defects. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We continued to use Drosophila as a model system to reveal the function of sialylation in animal nervous system. Our combined in vitro and in vivo approaches revealed that His 406 is a part of DSIAT catalytic site. We provided several lines of evidence suggesting that the neurological phenotype of DSiaT mutant is due to the effect on Para, a sodium voltage-gated channel. Our results suggested that Para is a direct target of DSiaT activity, which provides a mechanistic insight into the role of sialylation in the regulation of neuronal excitability. We discovered that downregulation of DSIAT expression in adult brains is a posttranscriptional mechanism possibly conserved in higher animals, including humans. We continued elucidating major players of the genetic and biochemical pathways of sialylation in Drosophila. These experiments revealed novel players involved in neuronal signaling and sialylation that are evolutionarily conserved from Drosophila to humans, including CSAS, GalNAcT-A and GalNAcT-B. Overall, our results shed light on evolutionary conserved mechanisms of neural-specific sialylation and suggested that similar processes are involved in the regulation of neural transmission in mammalian brain. The results of our experiments were presented at National and International conferences and Society meetings (Annual Conference of the Society for Glycobiology) The results were also disseminated via presentations and discussions during interactions with collaborators (one collaborative publication was published and one was submitted). We share our protocols and other research information with a broader scientific community via publications, conference presentations, and personal communication. We make all reagents generated in our research (such as DNA constructs, engineered mutant and transgenic flies, tissue culture lines, etc.) freely available to other researchers for further exploration of the involvement of sialylation in neural development. Two postdoctoral trainees have been mentored and one PhD student has graduated during reporting period. Currently, two postdoctoral research associates, two graduate students and a research technician are being mentored in the laboratory while actively participating in the project.
Publications
- Nakamura N, Stalnaker S, Lyalin D, Lavrova O, Wells L, Panin V. Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted. Glycobiology. 2010 20:381-94 [Epub 2009 Dec 7].
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: We have been successfully conducting the experiments described in our project, and the data obtained in these experiments have been thoroughly analyzed. Our project was concentrated on the following aims: 1. To characterize the biochemical activity and structure-function relationship of DSIAT protein. The structure-function relationship was analyzed by creating point mutants and examining their activity in cell culture and in vivo. Mutant forms revealed that even very low level of DSIAT activity is sufficient for normal CNS function. In addition, we found critical importance of H406 residue for catalytic activity of the protein. 2. To comprehensively characterize the expression pattern of the DSiaT gene during development, and to determine the cellular and subcellular distribution of DSIAT. 3. To characterize the mutant phenotype of DSiaT. The analysis of DSiaT mutant phenotypes has been mainly focused on the morphology and electrophysiology of larval NMJ and genetic interactions with genes potentially involved in DSiaT pathway, including known paralytic mutants, as well as novel genes predicted to affect sialylation through their involvement in the production of activated sugar donor, CMP-Sia. We continued to explore the genetic interaction between DSiaT and para, the gene encoding a subunit of sodium voltage-gated channel. We confirmed the specificity of detected interaction by rescue experiments with genetic duplication for para, as well as using a different para loss-of-function allele. We revisited the overexpression of sialidase in vivo using genotypes with increased level of ectopic expression in the CNS (by combining several transgenic constructs). These experiments resulted in locomotor abnormalities that partially mimic DSiaT mutant phenotype. This result is consistent with the conclusion that DSiaT phenotype is mainly due to the absence of sialylation within the CNS. Aim 4. To identify and characterize the in vivo molecular targets of DSIAT. We also continued the genetic interaction assays with genes potentially encoding sialylation targets and other molecules working in the same genetic pathway. This genetic interaction screen approach has been focused mostly on paralytic mutants. These experiments allowed us to focus our further investigation on para as a potential target of sialylation. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Using Drosophila as a model organism, we continued to utilize Drosophila model for elucidating the molecular and genetic mechanisms of sialylation in the development and functioning of animal nervous system. Our structure-function studies have revealed the crucial importance of His406 for sialyltransferase activity suggesting that it is a part of DSIAT catalytic site. We provided evidence that neurological DSiaT mutant phenotype is indeed due to the absence of sialyltransferase catalytic activity rather than the protein per se (e.g., ligand/substrate-binding activity). We discovered downregulation of DSIAT expression in adult brains suggesting a complex regulatory mechanism possibly conserved in higher animals. We started elucidating major players of the genetic and biochemical pathways of sialylation in Drosophila, which have revealed genes involved in neuronal signaling and sialylation that are evolutionarily conserved from Drosophila to humans. Our results reveal evolutionary conserved mechanisms that are likely evolutionary conserved and similarly underlie the regulation of human DSIAT homologue, ST6GAL II, at postembryonic stages in the brain. The results of our experiments were presented at National and International conferences and Society meetings (Annual Conference of the Society for Glycobiology) The results were also disseminated via presentations and discussions during invited seminars with collaborators (seminar in York University, Toronto, CA). We openly share our protocols and other research information with broader scientific community via publications, conference presentations, and personal communication. We make all reagents generated in our research (such as DNA constructs, engineered mutant and transgenic flies, tissue culture lines, etc.) freely available to other researchers for further exploration of the involvement of sialylation in neural development. Two postdoctoral trainees have been mentored during the project. Currently, one postdoc, two graduate students and a research technician are being mentored in the laboratory while actively participating in the project.
Publications
- Koles K, Repnikova E, Pavlova G, Korochkin LI, Panin VM. 2008. Sialylation in protostomes: a perspective from Drosophila genetics and biochemistry. Glycoconj J. Jun 21.
- Repnikova E., Koles K, Nakamura M., Tao C., Ambavane A.,Sullivan D., and Panin V. 2008. Function of sialyltransferase in Drosophila CNS. Glycobiology 18: 943.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: 1. One of the aims of the project was to characterize the glycosylation of Drosophila sialyltransferase protein, DSIAT, and its impact on the biochemical activity of the enzyme. We purified and several different deletion constructs and, in collaboration with Complex Carbohydrate Research Center (Athens, GA), initiated comprehensive analysis of their glycans by mass spectrometry. Two more constructs were made for bacterial expression system, and we will attempt to crystallize these protein constructs to obtain detailed structural information about this enzyme. 2. We continued to comprehensively characterize the expression pattern of the sialyltransferase gene (DSiaT) during development. We investigated the temporal profile of the gene's expression at different developmental stages. Our experiments revealed extremely dynamic expression pattern, with elevated expression that correlates with CNS remodeling stages, indicating a potential functional link to the CNS
plasticity. Early embryonic expression pattern was also characterized. We continue experiments on more precise mapping of expressing neurons with available markers, and plan to study in more detail subcellular localization of the protein in expressing neurons using immunostaining and confocal microscopy. Events: The results of our experiments were presented at National and International conferences and Society meetings (Annual Conference of the Society for Glycobiology, Annual Drosophila Conference of the Genetics Society of America, EMBO Workshop on Developmental Glycobiology (Lille, France)). The results were also disseminated via presentations and discussions during invited seminars with collaborators (seminars in Oxford University and in the Imperial College London, UK). Service: During the project, the PI's laboratory has been maintaining a microscopy imaging facility in the department that is open to the university research community, while Dr. Panin was responsible for regular
training of microscope users. Another aspect of professional service was the participation in activities of a broader research community. This involves reviewing research papers (ad hoc for BMC Developmental Biology, FEBS Journal, Genetics, JACS, JMB, Zoology, Electrophoresis), grant applications (ad hoc for the Austrian Science Fund (FWF) and the Natural Sciences and Engineering Research Council of Canada (NSERC)), and participation in grant peer review panels (NIH special emphasis panel "Alliance of Glycobiologists for Detection of Cancer and Cancer Risk", NCI, Bethesda, 2006; American Heart Association, Western Review Consortium, Houston, 2007), as well as in the Consortium for Functional Glycomics, a large research initiative funded by NIH (NIGMS) to define the paradigms of protein-carbohydrate interactions in cell communication. Dissemination of the results include outreach activities of visiting collaborators (CCRC, University of Georgia, Athens; Oxford University, Oxford, and
Imperial College London, UK), and establishing new collaborations within Texas A&M (Dr. Zoran's laboratory, Biology Department).
PARTICIPANTS: Research Personnel: Dr. Vlad Panin (PI), Dr. Kate Koles and Dr. Naosuke Nakamura (Postdoctoral Fellows); Elena Repnikova, Dmitry Lyalin, David Sullivan, Haiwen Li (graduate students); Ted Markulin, Caroline Tao, Apoorva Ambavane, Christina Ramos, Stacey Whitman (undergraduate student workers). Collaborations: Dr. Anne Dell (Imperial College, London, UK), Dr. Krylov, (York University, Toronto, CA), Dr. Tiemeyer, Dr. Wells, Dr. Azadi (CCRC, University of Georgia, Athens), Dr. Larry Dangott and Dr. M. Zoran (Texas A&M University
TARGET AUDIENCES: Scientists working in a broad field of modern Biology and Biomedical Sciences, including areas of Developmental Biology, Glycobiology, Biochemistry, Genetics, Neurobiology & Neurophysiology, Entomology. Audience also includes students and trainees who are studying and working in these areas.
Impacts
Using Drosophila as a model organism, we have elucidated one of the fundamental biological functions, the involvement of sialyltransferase in the developing and functioning of the central nervous system. Since Drosophila sialyltransferase represents the most ancient prototype of all animal sialyltransferases characterized to date, our findings suggest the presence of similar evolutionary conserved function of human sialyltransferases in the nervous system. This opens a new avenue of related research in mammalian organisms, which may shed light on molecular basis of human neurological abnormalities potentially associated with defects in sialylation pathway. The success of our research is significantly based on the expertise of the laboratory. For the most part, this expertise (in the form of developed protocols, reagents, and skills) was generated and accumulated in the course of the project. Additionally, the availability of the laboratory resources (such as grant
funds, equipment, genetic strains, etc) as well as the convenient access to the resources of the Department and University (commonly used equipment and facilities, service Centers and Laboratories, such as Microscopy Imaging Center, the Protein Chemistry Laboratory), all significantly contributed to the fruitful results of our experiments.
Publications
- Koles, K., Lim, J.-M, Aoki, K., Porterfield, M., Tiemeyer, M., Wells, L., and Panin, V.M.. 2007. Identification of N-glycosylated glycoproteins from the central nervous system of Drosophila melanogaster. Glycobiology. 17(12):1388-1403.
- Ishihara M, Lim J-M, Koles K, Panin V, Azadi P. 2007. Characterization of N-linked glycans on the Drosophila sialyltransferase protein, DSAIT, by mass spectrometry. Glycobiology 11: 1244.
- Koles K, Lim J-M, Aoki K, Porterfield M, Tiemeyer M, Wells L, Panin V. 2007. Identification of the major N-glycosylated glycoproteins from the fruit-fly brain. Glycobiology 11: 1257
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Progress 01/01/06 to 12/31/06
Outputs
1. One of the aims of the project was to characterize the glycosylation of Drosophila sialyltransferase protein, DSIAT, and its impact on the biochemical activity of the enzyme. We designed and tested four different deletion constructs and found that only one of them (including longer part of the stem region) is active and secreted. These experiments gave us important information about the requirement of different regions of sialyltransferase protein's stem domain for its activity. In addition, a new construct without protein A tag was made and tested in small-scale purification and in vitro assay experiments. We were able to obtain a shorter soluble version of DSIAT protein that still retains its enzymatic activity. This construct will be valuable for developing high-affinity antibody and structural studies of the DSIAT protein and its glycosylation. 2. We continued to comprehensively characterize the expression pattern of the sialyltransferase gene (DSiaT) during
development. We found that sialyltransferase is expressed in many motor neurons. We also identified a group of cholinergic interneurons that express DSiaT protein. In adults, we found that DSIAT is highly produced in the optic lobes, including lamina and medulla regions. In the central brain, the pattern of DSIAT expression was found to be restricted to a group of projection neurons that are implicated in delivering olfactory signals to the mushroom body. All these important findings established a new direction of phenotypic analyses aimed at characterization of potential morphological defects in the optic lobe of DSiaT mutants, as well as possible behavioral abnormalities associated with these defects. 3. We were also focusing on characterizing the mutant phenotype of DSiaT mutants at cellular, tissues, and organismal levels. We have completed analysis of temperature-induced paralysis of DSiaT mutants. We characterized novel KI48 mutant allele, the hypomorph that we generated using
gene targeting approach. This allele is valuable for our genetic interaction studies (aim 4). We also performed systematic analysis of viability, fertility, and longevity of DSiaT mutants. Our data indicate that DSiaT also plays an important role in oogenesis. In collaboration with Dr. Selleck (University of Minnesota), we revealed severe electrophysiological abnormalities of DSiaT mutants. The initial analysis of the fine morphology of adult visual system of DSiaT mutants using electron microscopy revealed signs of neuronal degeneration. 4. Finally, we studied genetic interactions between DSiaT and genes encoding its potential molecular targets. We found interactions with the putative protein O-fucosyltransferase-2 gene (Ofut2). We have recently purified from Drosophila brain the glycopeptide fraction which was enriched for sialylated structures using a series of lectin chromatography steps. We are currently collaborating with CCRC (University of Georgia) mass spectrometry experts on
characterizing this fraction. Our initial glycomic analysis of Drosophila embryos was recently published in collaboration with Dr. Haslam (Imperial College of London, UK).
Impacts
Our recent findings have shed light on the biological function of the Drosophila sialyltransferase, revealing its indispensable role in the nervous system development and physiology. Considering significant similarity and evolutionary conservation between the Drosophila sialyltransferase and its human counterparts, our results suggest similar involvement of these human enzymes in the corresponding physiological and molecular processes in the human organism and may explain molecular bases of some human neurological diseases.
Publications
- Lyalin, D. A., Koles, K., Roosendaal, S. D., Repnikova, E. A., Van Wechel, L., and Panin, V. M. (2006) The twisted gene encodes Drosophila protein O-mannosyltransferase 2 and genetically interacts with the rotated abdomen gene encoding Drosophila protein O-mannosyltransferase 1. Genetics. 172(1): 343-53.
- Luo Y, Koles K, Vorndam W, Haltiwanger RS, Panin VM. (2006) Protein O-fucosyltransferase 2 adds O-fucose to thrombospondin type 1 repeats. J. Biol. Chem. 281(14): 9393-9.
- North SJ, Koles K, Hembd C, Morris HR, Dell A, Panin VM, Haslam SM. (2006) Glycomic studies of Drosophila melanogaster embryos. Glycoconjugate Journal 23: 345-354.
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