1 Serveur Académique Lausannois SERVAL serval.unil.ch Author Manuscript Faculty of Biology and Medicine Publication This paper has been peer-reviewed but dos not include the final publisher proof-corrections or journal pagination. Published in final edited form as: Title: O-mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding. Authors: Yoshida-Moriguchi T, Yu L, Stalnaker SH, Davis S, Kunz S, Madson M, Oldstone MB, Schachter H, Wells L, Campbell KP Journal: Science (New York, N.Y.) Year: 2010 Jan 1 Volume: 327 Issue: 5961 Pages: DOI: /science In the absence of a copyright statement, users should assume that standard copyright protection applies, unless the article contains an explicit statement to the contrary. In case of doubt, contact the journal publisher to verify the copyright status of an article.
2 NIH Public Access Author Manuscript Published in final edited form as: Science January 1; 327(5961): doi: /science O-Mannosyl Phosphorylation of Alpha-Dystroglycan is Required for Laminin Binding Takako Yoshida-Moriguchi 1,2,3,4, Liping Yu 5, Stephanie H. Stalnaker 6, Sarah Davis 1,2,3,4, Stefan Kunz 7, Michael B.A. Oldstone 8, Harry Schachter 9, Lance Wells 6, and Kevin P. Campbell 1,2,3,4,* 1 Howard Hughes Medical Institute, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building, 285 Newton Road, Iowa City, Iowa , USA 2 Department of Molecular Physiology and Biophysics, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building, 285 Newton Road, Iowa City, Iowa , USA 3 Department of Neurology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building, 285 Newton Road, Iowa City, Iowa , USA 4 Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building, 285 Newton Road, Iowa City, Iowa , USA 5 Medical NMR Facility, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building, 285 Newton Road, Iowa City, Iowa , USA 6 Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Rd., Athens, Georgia 30602, USA 7 Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerland 8 Scripps Research Institute, Department of Immunology and Microbial Science, N. Torrey Pines Road, La Jolla, CA Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Abstract Alpha-dystroglycan is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-g domains and certain arenaviruses. Receptor binding is thought to be mediated by a post-translational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Here, using mass spectrometry- and NMR-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-dystroglycan, which was required for laminin binding. We demonstrated that patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy, as well as mice with myodystrophy, commonly have defects in a post-phosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the likeacetylglucosaminyltransferase (LARGE) protein. Our findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy. Diverse post-translational modifications influence the structure and function of many proteins. Dystroglycan (DG) is a membrane protein that requires extensive post-translational processing in order to function as an extracellular matrix receptor. It is comprised of an extracellular α- * To whom correspondence should be addressed. Supporting Online Material Materials and Methods Figs. S1 to S12 Table S1
3 Yoshida-Moriguchi et al. Page 2 DG subunit and a transmembrane β-dg subunit (1). α-dg serves as a receptor for extracellular matrix laminin G domain-containing ligands such as laminin (1) and agrin (2) in both muscle and brain, and these interactions depend on an unidentified post-translational α-dg modification. α-dg is also the cellular receptor for lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), and clade C New World arenaviruses (3,4). Although the binding sites for LCMV and LFV on α-dg have not yet been identified, they are thought to overlap with the modification recognized by laminin (5,6). Glycosyltransferase-mediated glycosylation is one form of post-translational modification that can modulate protein structure and function. The main forms in mammals are N- and O- glycosylation, and these are distinguished by how the oligosaccharide moiety links to the amino acid. Mutations in six known or putative glycosyltransferase genes POMT1 (7), POMT2 (8), POMGnT1 (9), fukutin (10), FKRP (11), and LARGE (12) have been identified in patients with congenital muscular dystrophy (CMD). These disorders cover a spectrum of abnormalities affecting the brain, eye, and skeletal muscle, and show a dramatic gradient of phenotypic severity ranging from the most devastating in Walker-Warburg syndrome (WWS; OMIM# ), to less severe in muscle-eye-brain disease (MEB; OMIM# ) and Fukuyama CMD (FCMD; OMIM# ), and to mild limb-girdle muscular dystrophies. In these diseases, the ability of α-dg to bind laminin is markedly reduced (13), suggesting that these (putative) glycosyltransferases participate in the post-translational modification that enables α-dg to bind laminin. Whereas the molecular functions of LARGE, fukutin, and FKRP remain unclear, POMT1/2 (14) and POMGnT1 (9) are known to catalyze two steps in the biosynthesis of an O-mannosyl tetrasaccharide (NeuNAc-α-2,3-Gal-β-1,4-GlcNAc-β-1,2-Man) that is found in high abundance on both brain and muscle α-dg (15,16). However, this glycan itself is not likely the laminin-binding moiety, since glycosidase-mediated removal of all or most of the three sugars at the non-reducing terminus does not reduce α-dg binding to laminin (17). To determine which post-translational modification is necessary for the α-dg/laminin interaction, we processed wheat germ agglutinin-enriched proteins (glycoproteins) from C57BL/6J (wild type, Wt) muscle using various enzymatic and chemical treatments. We then analyzed the products by immunoblotting with antibodies against α-dg core protein (CORE), the laminin-binding form of α-dg (IIH6), and β-dg core protein (AP83), as well as by laminin overlay assay. Treatment with cold aqueous hydrofluoric acid (HFaq), which specifically cleaves phosphoester linkages (18) resulted in reduction of the α-dg M r from 150- to 70-kDa, loss of IIH6 immunoreactivity and laminin binding (Fig. 1A), and loss of binding to LFV and LCMV (Fig. 1B). As the M r of N-glycosylated β-dg did not change (Fig. 1A), these effects were not caused by the degradation of either peptide or glycosyl linkages. A quantitative solidphase assay revealed that a 97% reduction in total high-affinity binding to laminin was achieved (Fig. 1C). HFaq treatment also abolished laminin-receptor activity of α-dg in heart, brain and kidney (fig. S1A). We next tested whether N-glycan and/or the two O-glycans known to modify the laminin-binding form of α-dg Core1 O-glycan and the O-mannosyl tetrasaccharide (in either the sialylated or fucosylated form) (15,16) are sensitive to HFaq treatment. Immunoblotting of Wt-muscle glycoproteins treated with several cocktails of glycosidases that degrade these three glycans showed that the glycosidase-mediated reduction in α-dg glycosylation was impervious to HFaq treatment (Fig. 1D). Similar experiment using muscle glycoproteins from the CMD-model mouse LARGE myd, in which a mutation in LARGE prevents the α-dg modification necessary for laminin-binding (19), revealed that HFaq treatment did not significantly reduce the M r of α-dg (Fig. 1D). Thus, HFaq degrades specifically the laminin-binding moiety on α-dg. Finally, we tested the type of phosphorylation involved by digesting Wt-muscle glycoproteins with alkaline phosphatase. The laminin-receptor activity of α-dg (fig. S1B) was not affected, suggesting that functional modification of α-dg involves an internal phosphoryl linkage rather than a monoester-linked phosphate.
4 Yoshida-Moriguchi et al. Page 3 To verify that α-dg is phosphorylated, we labeled human embryonic kidney cells (HEK293) expressing Fc-tagged α-dg recombinants (Fig. 2A) that are secreted into the medium with [ 32 P]-orthophosphate. Phosphor-imaging showed that secreted DGFc4, which contains only the mucin-like region of α-dg owing to cleavage of its N-terminus by a furin-like protease (20), was phosphorylated (Fig. 2B). When [ 32 P]-DGFc4 was hydrolyzed under conditions conducive to the dissolution of polypeptide and phosphoester linkages to carbohydrates, but not to linkages to amino acids (21), inorganic phosphate but not phospho-amino acids were generated (fig. S2), suggesting that phosphorylation does not occur directly on the peptide. To test if the phosphorylation depends on glycosylation, we expressed DGFc5 in [ 32 P]- orthophosphate-labeled human cells derived from POMT1-mutated WWS, POMGnT1- mutated MEB, fukutin-mutated FCMD, and control cells, as well as in fibroblasts from Large myd and Wt mice (Fig. 2C). All except the POMT1-mutated WWS cells secreted [ 32 P]- phosphorylated DGFc5 into the medium, strongly suggesting that phosphorylation occurs on the O-linked mannose of α-dg. By measuring inorganic phosphate following acid hydrolysis, we confirmed that native α-dg purified from rabbit skeletal muscle is phosphorylated, and further quantitated this modification at 4.7 mol phosphate per mol protein (SEM=0.22, n=3). To assess if CMD cells that synthesize phosphorylated α-dg can further modify the phosphate residue, we immunoprecipitated glycoproteins from mouse Large myd and Wt muscle, as well as from human POMGnT1-mutated MEB, fukutin-mutated FCMD, and control muscle, using immobilized metal affinity chromatography (IMAC)-beads that bind to monoester-linked, but not diester-linked, phosphorylated compounds. Only fukutin-mutated FCMD and Large myd muscle α-dg were captured by the beads, revealing that the phosphate residue does not undergo further modification in these CMD cells (Fig. 2D). This suggests that fukutin and LARGE participate in a common pathway to assemble the laminin-binding moiety onto the phosphorylated O-linked mannose. This speculation is compatible with the fact that α-dg prepared from Large myd muscle rescued by adenovirus-mediated expression of LARGE regains laminin-receptor activity and concomitantly loses its affinity for IMAC beads (fig. S3). In the case of POMGnT1-mutated MEB patient muscle, several forms of α-dg were observed; the majority of these were captured by the beads, although a certain amount of α-dg with laminin-binding activity was detected in the void fraction (Fig. 2D). This finding suggests that a defect in POMGnT1 partially inhibits modification on the phosphoryl branch chain of the O- mannosyl glycan on α-dg. DGFc4 produced by HEK293 cells bound to IMAC-beads (fig. S4A) and gained lamininbinding activity when it was co-expressed with LARGE (fig. S4B); such a gain in activity has also been observed in FCMD, MEB, and Large myd cells, both in this study and elsewhere (22). To determine the structure of the phosphorylated O-mannosyl glycan that appears to be necessary to assemble the laminin-binding moiety, we prepared O-glycans from HEK293- expressed DGFc4 by reductive β-elimination, and isolated the phosphorylated O-glycan using IMAC-beads. LTQ mass spectrometry-based analyses detected prominent ions at m/z 667 ([M- H] ) and m/z 333 ([M-2H] 2 ) which are assigned as a phosphorylated trisaccharide composed of HexNAc 2 Hexitol 1 (fig. S5), and analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed the compositional sugars to be GlcNAc, GalNAc, and mannitol (fig. S6). Homo- and heteronuclear NMR techniques were used to assign the 13 C/ 1 H HMQC spectrum of the reduced O- glycan (Fig. 3A). The GlcNAc (subunit B) was assigned using DQF-COSY and TOCSY spectra with a series of mixing times (fig. S7). The GalNAc (subunit C) was partially assigned based on a selective-tocsy-hsqc spectrum (Fig. 3B). The GalNAc (subunit C) is linked via a β1-3 linkage to the C3 position on GlcNAc (subunit B), which is in turn connected via a β1 4 linkage to the C4 position on mannitol (subunit A), as evidenced by the observed HMBC cross peaks CH1/BC3 and BH1/AC4 (Fig. 3C). The phosphate group is attached to the C6 position of the mannitol (subunit A) as determined from the cross peaks 31 P/AH6 and 31 P/
5 Yoshida-Moriguchi et al. Page 4 A6H detected in the 31 P/ 1 H COSY spectrum (Fig. 3D). The complete NMR resonance assignments of the reduced O-glycan and its inter-residue correlations detected in the NOESY and HMBC spectra are summarized in table S1, and the determined structure is shown in Fig. 3E. To verify that this phosphorylated trisaccharide modifies the mucin-like domain of DGFc4, we enriched the trypsinized peptides using Wisteria Floribunda agglutinin-lectin and analyzed the GalNAc-terminated peptides by LCMS/MS. MS/MS fragmentation patterns at m/z (Fig. 4A), (fig. S9), and (fig. S10) identified a peptide (amino acids 374 to 389 of α-dg; Accession ID: CAA45732) bearing these modifications the phosphorylated trisaccharide in conjunction with Hex-HexNAc-Hex, HexNAc-Hex or Hex. The presence of non-phosphorylated mannose-initiated structures on y3, y4, and y10 ions revealed that Thr 379 is modified by the phosphorylated trisaccharide in all cases (Figs. 4AB, S9, and S10). To assess where within the cell α-dg is phosphorylated, we separated the ER and Golgi of DGFc4-overexpressing HEK293 cells using an iodixanol gradient (fig. S11A). Immunoprecipitation with IMAC-beads showed that only the Golgi fraction contained phosphorylated DGFc4, suggesting that phosphorylation occurs in the Golgi (fig. S11B). Mannose-6-phosphorylation is known to occur on N-glycans during their passage through the Golgi, and to serve as the marker for targeting newly synthesized lysosomal enzymes to the lysosome (23). In this mannose-6-phosphate synthetic pathway, mannose is phosphorylated by the sequential actions of two enzymes: UDP-GlcNAc:lysosomal enzyme GlcNAc-1- phosphotransferase (OMIM# ), which transfers GlcNAc-1-phosphate to the C6 hydroxyl of mannose; and GlcNAc-1-phosphodiester α-n-acetylglucosaminidase (OMIM# ), which removes the terminal GlcNAc to form mannose-6-phosphate. Analysis of fibroblasts derived from patients with Mucolipidosis II (OMIM ), which have a defect in GlcNAc-1-phosphotransferase, revealed that they can synthesize the laminin-binding form of α-dg (Fig. 4C). Thus, an unidentified pathway phosphorylates the C6 hydroxyl of the O- mannosyl glycan. We have identified a phosphorylated O-glycan on recombinant α-dg synthesized in HEK293 cells. Further, our comparative studies of α-dg in CMD and control muscle reveal that the phosphoryl chain on O-mannosyl glycan is required to assemble the laminin-binding moiety, and that LARGE directly participates in the post-phosphorylation biosynthetic pathway. MEB, FCMD and Large myd cells, which have genetically distinct abnormalities, nevertheless show a similar defect in processing of the phosphoryl branch chain on the O-mannosyl glycan (Fig. 4D). These convergent mechanisms to pathology offer an explanation for previous reports that forced expression of LARGE can circumvent defects in α-dg modification in these CMD cells (22). We have shown that overexpressing LARGE, a putative glycosyltransferase with catalytic domains sharing homology with β-1,3-n-acetylglucosaminyltransferase and bacterial glycosyltransferase (19), increases the affinity of the cell surface for both the IIH6 antibody (Fig.S12A) and the Vicia villosa lectin (Fig. S12B). Thus, LARGE is likely to transfer glycosyl moiety to an O-linked mannose-6-phosphate. To our knowledge, we provide the first evidence that a vertebrate non-gpi-anchored glycoprotein is modified by a phosphodiester linkage. Glycoproteins in the cell walls of yeasts and fungi bear phosphodiester-linked glycans generated by a process involving phosphorylation on the C6 hydroxyl of mannose (24). It is notable that α-dg, which is well conserved as an epithelial cell-surface protein in species ranging from lower vertebrates to mammals, is likewise modified by this ancient type of glycosylation. A recent study has shown that the most severe form of CMD WWS is a genetically heterogeneous disease. Moreover, only 40% of WWS cases are explained by mutations in known CMD-causative genes (24). Thus, a defect in the phosphorylation of an O-linked mannose may be responsible for severe CMD in which case the discovery of mutations in novel genes responsible for WWS may not be far off.
6 Yoshida-Moriguchi et al. Page 5 Supplementary Material Acknowledgments Refer to Web version on PubMed Central for supplementary material. 25. We thank Michael Madson for insightful suggestions; M. E. Anderson and D. Venzke for technical assistance; L. M. Teesch for mass spectrometry, I. Nishino for providing WWS-patient cells, Paul D. Wellstone Muscular Dystrophy Cooperative Research Center Core B for providing patient cells and biopsies; the Gene Transfer Vector Core (UI, supported by NIH/NIDDK P30 DK 54759) for generating adenoviruses; the Glycotechnology Core Resource (UCSD) for HPAEC-PAD analysis; the Integrated Technology Resource for Biomedical Glycomics (UGA, supported by NIH/ NCRR P41 RR018502) for tandem mass spectrometry; and past and present members of the Campbell lab for scientific contributions. This study was supported in part by a Paul D. Wellstone Muscular Dystrophy Cooperative Research Center Grant (1U54NS053672, K.P.C.), a U.S. Department of Defense Grant (W81XWH , K.P.C.), and AI55540 (to S.K. and M.B.A.O.). K.P.C. is an Investigator of the Howard Hughes Medical Institute. References and Notes 1. Ibraghimov-Beskrovnaya O, et al. Nature 1992;355:696. [PubMed: ] 2. Gee SH, Montanaro F, Lindenbaum MH, Carbonetto S. Cell 1994;77:675. [PubMed: ] 3. Cao W, et al. Science 1998;282:2079. [PubMed: ] 4. Spiropoulou CF, Kunz S, Rollin PE, Campbell KP, Oldstone MB. J Virol 2002;76:5140. [PubMed: ] 5. Kunz S, Sevilla N, McGavern DB, Campbell KP, Oldstone MB. J Cell Biol 2001;155:301. [PubMed: ] 6. Kunz S, Rojek JM, Perez M, Spiropoulou CF, Oldstone MB. J Virol 2005;79:5979. [PubMed: ] 7. Beltran-Valero de Bernabe D, et al. Am J Hum Genet 2002;71:1033. [PubMed: ] 8. van Reeuwijk J, et al. J Med Genet 2005;42:907. [PubMed: ] 9. Yoshida A, et al. Dev Cell 2001;1:717. [PubMed: ] 10. Kobayashi K, et al. Nature 1998;394:388. [PubMed: ] 11. Brockington M, et al. Am J Hum Genet 2001;69:1198. [PubMed: ] 12. Longman C, et al. Hum Mol Genet 2003;12:2853. [PubMed: ] 13. Michele DE, et al. Nature 2002;418:417. [PubMed: ] 14. Manya H, et al. Proc Natl Acad Sci U S A 2004;101:500. [PubMed: ] 15. Sasaki T, et al. Biochim Biophys Acta 1998;1425:599. [PubMed: ] 16. Chiba A, et al. J Biol Chem 1997;272:2156. [PubMed: ] 17. Combs AC, Ervasti JM. Biochem J 2005;390:303. [PubMed: ] 18. Ilg T, et al. J Biol Chem 1996;271: [PubMed: ] 19. Grewal PK, Holzfeind PJ, Bittner RE, Hewitt JE. Nat Genet 2001;28:151. [PubMed: ] 20. Kanagawa M, et al. Cell 2004;117:953. [PubMed: ] 21. Ilg T, et al. J Biol Chem 1994;269: [PubMed: ] 22. Barresi R, et al. Nat Med 2004;10:696. [PubMed: ] 23. Dahms NM, Lobel P, Kornfeld S. J Biol Chem 1989;264: [PubMed: ] 24. Manzini MC, et al. Hum Mutat 2008;29:E231. [PubMed: ]
7 Yoshida-Moriguchi et al. Page 6 Fig. 1. Chemical dephosphorylation by HFaq treatment abolishes laminin and virus binding to α-dg in tissues from Wt mice. (A and B) Treated glycoproteins prepared from Wt muscle were subjected to: (A) immunoblotting with antibodies against the α-dg core protein (CORE) or the laminin-binding form α-dg epitope (IIH6) and β-dg (AP83), or to laminin overlay assay; (B) virus overlay assays with γ-inactivated LFV or LCMV cl-13. (C) Wt muscle glycoproteins with and without HFaq-treatment were subjected to a solid-phase laminin-binding assay (n=3). Open circles=treated; closed circles=untreated. Error bars indicate standard deviation. (D) Muscle glycoproteins from Wt and Large myd (Myd) mice were digested using cocktails of glycosidases that degrade sialylated and/or fucosylated N-glycan, Core1 O-glycan, and O- mannosyl glycan, before (first 4 lanes) and after (last 4 lanes) HFaq treatment. The products were subjected to either immunoblotting with CORE antibody or laminin overlay assay.
8 Yoshida-Moriguchi et al. Page 7 Fig. 2. The mucin-like region of α-dg is phosphorylated in an O-mannosylation-dependent manner. (A) Structures of recombinant α-dg constructs used in the study. (B and C) [ 32 P]- orthophosphate labeling of (B) Fc-Ctrl- or DGFc4-expressing HEK293 cells and (C) DGFc5- expressing cultured cells from CMD patients (WWS, MEB, FCMD) and control humans, and from Wt and Large myd (Myd) mice. Fc-tagged recombinant α-dg was isolated from the culture medium using protein-a agarose, separated by SDS-PAGE, stained with Coomassie brilliant blue (CBB) and analyzed by phosphor-imaging ([ 32 P]). Phosphorylation on α-dg required prior O-mannosylation. Asterisks indicate contaminating proteins derived from FBS. (D) IMAC-binding assay testing glycoproteins from Wt and Large myd mice, and from FCMD, MEB, and control human muscle (SkM).
9 Yoshida-Moriguchi et al. Page 8 Fig. 3. NMR analysis of phosphorylated O-glycan on HEK293-produced DGFc4. (A) HMQC spectrum where the assigned cross peaks are labeled with a letter for the subunit designated in (E), and a number for the position on that subunit. The folded cross peaks are indicated in red, and the cross peaks derived from sample impurities are marked by asterisks. (B) TOCSY- HSQC spectrum obtained using a selective excitation pulse at the subunit CH1 proton and a selective TOCSY mixing time of 113 ms. (C) HMBC (green) and HMQC (black) spectra for the assignment of interglycoside linkages. (D) 31 P/ 1 H COSY spectrum. (E) Structure of the O-glycan, with the sugar subunits labelled A C.
10 Yoshida-Moriguchi et al. Page 9 Fig. 4. Mapping of phosphorylated trisaccharide on HEK293-produced DGFc4, and characterization of mannosyl phosphorylation. (A) CID-MS/MS spectra from m/z (upper panel) and m/z (lower panel), revealing neutral loss pattern (upper panel) and peptide-derived b and y ions (lower panel) of the selected precursor ions at m/z Full FT mass spectrum is shown in fig. S8. Square: HexNAc; circle: Hexose. (B) Peptide and mononsaccharide unit identification based on fragmentation of the phosphorylated glycopeptide. (C) Glycoproteins prepared from cell lysates of fibroblasts derived from Mucolipidosis II patients and subjected to immunoblotting with CORE antibody, and to laminin overlay assay. (D) Schematic representation of O-mannosyl glycans on α-dg, with proteins involved in its biosynthesis, and diseases in which relevant steps are defective indicated. Shading indicates glycan identified in brain and muscle α-dg.
Glycobiology vol. 22 no. 5 pp. 662 675, 2012 doi:10.1093/glycob/cws002 Advance Access publication on January 11, 2012 Glycoproteomic characterization of recombinant mouse α-dystroglycan Rebecca Harrison
Mechanisms of Disease: congenital muscular dystrophies glycosylation takes center stage Paul T Martin SUMMARY Recent studies have defined a group of muscular dystrophies, now termed the dystroglycanopathies,
Secretion and Endocytosis Peter Takizawa Cell Biology Vesicular transport between organelles Glycosylation Protein sorting in the Golgi Endocytosis Secretory pathway delivers proteins and lipids to plasma
PO-CON1347E Structural Elucidation of N-glycans Originating From Ovarian Cancer Cells Using High-Vacuum MALDI Mass Spectrometry ASMS 2013 TP-708 Matthew S. F. Choo 1,3 ; Roberto Castangia 2 ; Matthew E.
Antigen Receptor Structures October 14, 2016 Ram Savan firstname.lastname@example.org 441 Lecture #8 Slide 1 of 28 Three lectures on antigen receptors Part 1 (Today): Structural features of the BCR and TCR Janeway Chapter
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
Study of different types of ubiquitination Rudi Beyaert (email@example.com) VIB UGent Center for Inflammation Research Ghent, Belgium VIB Training Novel Proteomics Tools: Identifying PTMs October
GlycoProfile II Enzymatic In-Solution N-Deglycosylation Kit Product Code PP0201 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description Glycosylation is one of the most common posttranslational
2/7/17 Glycomics & Glycoproteomics Lance Wells, omplex arbohydrate Research enter, Department of Biochemistry & Molecular Biology, and hemistry University of Georgia firstname.lastname@example.org NIGMS Biomedical
Enzymatic Protein Deglycosylation Kit Catalog Number EDEGLY Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description The EDEGLY kit contains all the enzymes and reagents needed to completely remove
Posttranslational modification of proteins Stephen Barnes, PhD email@example.com General classes of modification Biochemical event involving peptide processing Biochemical event stimulated by enzymes Chemical
Chapter 6 Antigen Presentation to T lymphocytes Generation of T-cell Receptor Ligands T cells only recognize Ags displayed on cell surfaces These Ags may be derived from pathogens that replicate within
Enhancing the baculovirus expression system with VANKYRIN technology Kendra Steele, Ph.D. Insect cells Can express mammalian proteins Similarities with mammalian cells Eukaryotic systems How protein are
Why would you want to study proteins associated with viruses or virus infection? Receptors Mechanism of uncoating How is gene expression carried out, exclusively by viral enzymes? Gene expression phases?
1. Which feature is true for signal sequences and for stop transfer transmembrane domains (4 pts)? A. They are both 20 hydrophobic amino acids long. B. They are both found at the N-terminus of the protein.
Flow-Through Electron Capture Dissociation in a novel Branched RF Ion Trap Takashi Baba, J. Larry Campbell, Yves Le Blanc, Jim. W. Hager and Bruce A. Thomson ASMS, June 18 / 2014 1 2014 AB SCIEX Trapping
MBB 694:407, 115:511 First Test Severinov/Deis Tue. Sep. 30, 2003 Name Index number (not SSN) Row Letter Seat Number This exam consists of two parts. Part I is multiple choice. Each of these 25 questions
Table S1. Sequence of human and mouse primers used for RT-qPCR measurements. Ca9, carbonic anhydrase IX; Ndrg1, N-myc downstream regulated gene 1; L28, ribosomal protein L28; Hif1a, hypoxia inducible factor
Exam 3 Fall 2015 Dr. Stone 8:00 Name There are 106 possible points (6 bonus points) on this exam. There are 8 pages. v o = V max x [S] k cat = kt e - ΔG /RT V max = k cat x E t ΔG = -RT lnk eq K m + [S]
AD Award Number: W81XWH-12-1-0248 TITLE: Targeted Inhibition of Tyrosine Kinase-Mediated Epigenetic Alterations to Prevent Resurgence of Castration-Resistant Prostate Cancer PRINCIPAL INVESTIGATOR: Dr.
Biological Mass Spectrometry April 30, 2014 Mass Spectrometry Has become the method of choice for precise protein and nucleic acid mass determination in a very wide mass range peptide and nucleotide sequencing
a e Na V 1.5 Ad-LacZ Ad- 110KD b Scn5a/ (relative to Ad-LacZ) f 150 100 50 0 p = 0.65 Ad-LacZ Ad- c Heart Lung Kidney Spleen 110KD d fl/fl c -/- DAPI 20 µm Na v 1.5 250KD fl/fl Rabbit IgG DAPI fl/fl Mouse
Different MHC alleles confer different functional properties on the adaptive immune system by specifying molecules that have different peptide binding abilities Location of MHC class I pockets termed B
Why chaperone vectors? A protein folding initiative An open discussion with structural biologists Protein Structure Initiative: Pilot Phase Whether the pilot phase achieved its goal depends on how we measure
Chapter 5: Amino Acids, Peptides & Proteins Amino acids share common functional groups Amino, Carboxyl & H bonded to C Distinct chemistry of s result of side chains Proteins & polypeptides are polymers
1of5 ELECTRONIC LETTER Mutations in the FKRP gene can cause muscle-eye-brain disease and Walker Warburg syndrome D Beltran-Valero de Bernabé, T Voit, C Longman, A Steinbrecher, V Straub, Y Yuva, R Herrmann,
SUPPLEMENTARY INFORMATION doi:1.138/nature9814 a A SHARPIN FL B SHARPIN ΔNZF C SHARPIN T38L, F39V b His-SHARPIN FL -1xUb -2xUb -4xUb α-his c Linear 4xUb -SHARPIN FL -SHARPIN TF_LV -SHARPINΔNZF -SHARPIN
Amino Acids 20 common amino acids there are others found naturally but much less frequently Common structure for amino acid COOH, -NH 2, H and R functional groups all attached to the a carbon Ionization
HIGH RESOLUTION MASS SPECTROMETRY (HRMS) IN DISCOVERY PROTEOMICS A clinical proteomics perspective Michael L. Merchant, PhD School of Medicine, University of Louisville Louisville, KY Learning Objectives
Supplementary Figures Supplementary Figure 1. Comparative analysis of thermal denaturation of enzymatically synthesized polyubiquitin chains of different length. a, Differential scanning calorimetry traces
Lipids ot different than other organic small molecules Carbohydrates Polymers of monosaccharides linked via glycosidic bonds (acetals/ ketals) many different combinationsvery interesting no time ucleic
Integrin v 3 targeted therapy for Kaposi s sarcoma with an in vitro evolved antibody 1 CHRISTOPH RADER, 2 MIKHAIL POPKOV, JOHN A. NEVES, AND CARLOS F. BARBAS III 2 Department of Molecular Biology and The
Phosphoproteome dynamics of Saccharomyces cerevisiae under heat shock and cold stress Evgeny Kanshin 1,5, Peter Kubiniok 1,2,5, Yogitha Thattikota 1,3, Damien D Amours 1,3 and Pierre Thibault 1,2,4 * 1
PREMIER Biosoft SimGlycan A high-throughput glycan and glycopeptide data analysis tool for LC-, MALDI-, ESI- Mass Spectrometry workflows SimGlycan software processes and interprets the MS/MS and higher
BIOSYNTHESIS OF CANCER-RELATED CARBOHYDRATE ANTIGENS Fabio Dall Olio Department of Experimental Pathology University of Bologna, Italy TOPICS OF THE LECTURE 1. Structure and function of some representative
Chemical Composition of the Cell B. Balen Table 2-2 Molecular Biology of the Cell ( Garland Science 2008) 1. Water the most abundant substance in the cell! Where did it come from? several hypothesis: -
BabyBio IMAC columns DATA SHEET DS 45 655 010 BabyBio columns for Immobilized Metal Ion Affinity Chromatography (IMAC) are ready-to-use for quick and easy purification of polyhistidine-tagged (His-tagged)
ame: TF: Section Time Life Science 1A Final Exam January 19, 2006 Please write legibly in the space provided below each question. You may not use calculators on this exam. We prefer that you use non-erasable
Enzymes Part III: regulation II Dr. Mamoun Ahram Summer, 2017 Advantage This is a major mechanism for rapid and transient regulation of enzyme activity. A most common mechanism is enzyme phosphorylation
Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic components Ionization source Mass analyzer Detector 1 Principles of Mass Spectrometry Proteins are separated by mass to charge ratio (limit
Lecture 13 Protein regulation Protein motion Antoine van Oijen BCMP201 Spring 2008 04/02 Section IV 04/09 Hands-on methods session / PS 4 due 1 Today s lecture 1) Mechanisms of protein regulation 2) Molecular
Supplementary Materials Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS Daryl A. Bosco, Gerardo Morfini, Murat Karabacak, Yuyu Song, Francois Gros-Louis,
A systematic investigation of CID Q-TO-MS/MS collision energies A systematic investigation of CID Q-TO-MS/MS collision energies to allow N- and O-glycopeptide identification by LC-MS/MS Abstract The MS
Allergy and Immunology Review Corner: Chapter 3, Part A (pages 37-45) of Cellular and Molecular Immunology (Seventh Edition), by Abul K. Abbas, Andrew H. Lichtman and Shiv Pillai. Chapter 3, Part A (Pages
Protein Reports CPTAC Common Data Analysis Pipeline (CDAP) v. 05/03/2016 Summary The purpose of this document is to describe the protein reports generated as part of the CPTAC Common Data Analysis Pipeline
AMINO ACIDS STRUCTURE, CLASSIFICATION, PROPERTIES. PRIMARY STRUCTURE OF PROTEINS Elena Rivneac PhD, Associate Professor Department of Biochemistry and Clinical Biochemistry State University of Medicine
Modeling Life s Important Compounds AP Biology CLASS SET OBJECTIVES: Upon completion of this activity, you will be able to: Explain the connection between the sequence and the subcomponents of a biological
Proteins? Protein function Protein folding Protein folding diseases Protein interactions Macromolecular assemblies The end product of Genes Protein Unfolding DOD Acid Catalysis DOD HDOD + N H N D C N C
LECTURE: 07 Title: IMMUNE CELL SURFACE RECEPTORS AND THEIR FUNCTIONS LEARNING OBJECTIVES: The student should be able to: The chemical nature of the cellular surface receptors. Define the location of the
MICR2209 Structure and Function of Antigen Recognition Molecules Dr Allison Imrie firstname.lastname@example.org 1 Synopsis: In this lecture we will examine the major receptors used by cells of the innate and
Protein Degradation Cellular functions of protein degradation 1. Elimination of misfolded and damaged proteins: Environmental toxins, translation errors and genetic mutations can damage proteins. Misfolded
Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis Ying Qing Yu Waters Corporation, Milford, MA, USA APPLICATION BENEFITS
Analysis of Uncomplexed and Copper-complexed Methanobactin with UV/Visible Spectrophotometry, Mass Spectrometry and NMR Spectrometry Lee Behling, Alan DiSpirito, Scott Hartsel, Larry Masterson, Gianluigi
CF@LANTA RDP Cores Highlights: the CF Analytics Core Facundo M. Fernández School of Chemistry and Biochemistry Georgia Institute of Technology CF@LANTA RDP Center The CF@LANTA RDP Center at Emory University
Mass spectrometry in glycomics research: Application to IgA nephropathy Part I Jan Novak, Ph.D. and Matthew B. Renfrow, Ph.D. In: Proteomics and mass spectrometry 2007 March 9, 2007 IgA Nephropathy The
Comparison of Relative Quantification of Monoclonal ntibody N-glycans Using Fluorescence and MS Detection pplication Note iotherapeutics & iologics uthors scar Potter and Gregory Staples gilent Technologies,
Molecular Graphics Perspective of Protein Structure and Function VMD Highlights > 20,000 registered Users Platforms: Unix (16 builds) Windows MacOS X Display of large biomolecules and simulation trajectories
AP Biology Name: Block Chapter 3 Guided Reading Notes Carbon and the Molecular Diversity of Life Most of this chapter is new material. We will discuss it all in detail. Section 1 1. Make an electron distribution
Proteins: Proteomics & Protein-Protein Interactions Part I Jesse Rinehart, PhD Department of Cellular & Molecular Physiology Systems Biology Institute DNA RNA PROTEIN DNA RNA PROTEIN Proteins: Proteomics
REGULATION OF ENZYME ACTIVITY Medical Biochemistry, Lecture 25 Lecture 25, Outline General properties of enzyme regulation Regulation of enzyme concentrations Allosteric enzymes and feedback inhibition
MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel Friday 11/12/04 7.012 Quiz 3 Answers A > 85 B 72-84
Brefeldin prevents assembly of the coats required for budding Nocodazole disrupts microtubules Constitutive: coatomer-coated Selected: clathrin-coated The formation of synaptic vesicles Nerve cells (and
Endoplasmic Reticulum What s ER? How is ER? Why is ER? definition description functions Nissl s bodies neurons Berg s bodies hepatocytes Organelle structure histocytochemical evidences Ergastoplasm pancreatic
Wolff-Parkinson-White Syndrome and PRKAG2 Maggie Beatka University of Wisconsin-Madison http://www.beatmap.net/portfolio-detail/human-cardiovascular-system-3drenderings/ What causes Wolff-Parkinson-White?
CHAPTER 8 Major Histocompatibility Complex (MHC) What is is MHC? HLA H-2 Minor histocompatibility antigens Peter Gorer & George Sneell (1940) Significance of the MHC role in immune response role in organ
OpenStax-CNX module: m44532 1 RNA Processing in Eukaryotes * OpenStax This work is produced by OpenStax-CNX and licensed under the Creative Commons Attribution License 3.0 By the end of this section, you
A Fully Integrated Workflow for LC-MS/MS Analysis of Labeled and Native N-Linked Glycans Released From Proteins Udayanath Aich, 1 Julian Saba, 2 Xiaodong Liu, 1 Srinivasa Rao, 1 Yury Agroskin, 1 and Chris
Introduction to Peptide equencing Quadrupole Ion Traps tructural Biophysics Course December 3, 2014 12/8/14 Introduction to Peptide equencing - athan Yates 1 Why are ion traps used to sequence peptides?
Chapter 6 Enzymes: The Catalysts of Life Lectures by Kathleen Fitzpatrick Simon Fraser University Activation Energy and the Metastable State Many thermodynamically feasible reactions in a cell that could
Advantages of Ion Mobility Q-TOF for Characterization of Diverse Biological Molecules Add a New Dimension to your Research Capability with Agilent s New Drift Ion Mobility Q-TOF System Overview: 6560 IM
Cell Structure 1 Don t Freak Out Test on cell organelle on Friday! This test should be a buffer test and help raise your overall test score. All information will come from this week! 2 Cells Provide Compartments
Main differences between plant and animal cells: Plant cells have: cell walls, a large central vacuole, plastids and turgor pressure. The Cell Wall The primary cell wall is capable of rapid expansion during
Main differences between plant and animal cells: Plant cells have: cell walls, a large central vacuole, plastids and turgor pressure. Animal cells have a lysosome (related to vacuole) and centrioles (function
OpenStax-CNX module: m44452 1 Propagation of the Signal OpenStax College This work is produced by OpenStax-CNX and licensed under the Creative Commons Attribution License 3.0 By the end of this section,
RESEARCH FUND FOR THE CONTROL OF INFECTIOUS DISEASES Liver specific glycoforms of serum proteins in chronic hepatitis B infection: identification by lectin affinity chromatography and quantitative proteomic
Probing the catalytic mechanism of an antifibrotic copper metallodrug 4 December 2017 Lizelle Lubbe Supervisor: Prof Ed Sturrock Transition metals offer benefit of designing compounds with complex architectures,