Skip to main content
SpringerLink
Log in

Glycosylation of Type I Collagen

  • Protocol
  • First Online:
Post-Translational Modification of Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1934))

Abstract

Fibrillar type I collagen is the most abundant structural protein in most tissues and organs. One of the unique and functionally important characteristics of collagen is sequential posttranslational modifications of lysine (Lys) residues. In the endoplasmic reticulum, hydroxylation of specific Lys occurs producing 5-hydroxylysine (Hyl). Then, to the 5-hydroxyl group of Hyl, a single galactose unit can be attached to form galactosyl-Hyl (Gal-Hyl) and further glucose can be added to Gal-Hyl to form glucosylgalactosyl-Hyl (GlcGal-Hyl). These are the only two O-linked glycosides found in mature type I collagen. It has been shown that this modification is critically involved in a number of biological and pathological processes likely through its regulatory roles in collagen fibrillogenesis, intermolecular cross-linking, and collagen-cell interaction. Recently, with the advances in molecular/cell biology and analytical chemistry, the molecular mechanisms of collagen glycosylation have been gradually deciphered, and the type and extent of glycosylation at the specific molecular loci can now be quantitatively analyzed. In this chapter, we describe quantitative analysis of collagen glycosylation by high-performance liquid chromatography (HPLC) and semiquantitative, site-specific analysis by HPLC-tandem mass spectrometry.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ishikawa Y, Wirz J, Vranka JA, Nagata K, Bachinger HP (2009) Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex. J Biol Chem 284:17641–17647

    Article  CAS  Google Scholar 

  2. Weis MA, Hudson DM, Kim L, Scott M, Wu JJ, Eyre DR (2010) Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly. J Biol Chem 285:2580–2590

    Article  CAS  Google Scholar 

  3. Terajima M, Taga Y, Chen Y, Cabral WA, Hou-Fu G, Srisawasdi S, Nagasawa M, Sumida N, Hattori S, Kurie JM, Marini JC, Yamauchi M (2016) Cyclophilin-B modulates collagen cross-linking by differentially affecting lysine hydroxylation in the helical and telopeptidyl domains of tendon type I collagen. J Biol Chem 291:9501–9512

    Article  CAS  Google Scholar 

  4. Heard ME, Besio R, Weis M, Rai J, Hudson DM, Dimori M, Zimmerman SM, Kamykowski JA, Hogue WR, Swain FL, Burdine MS, Mackintosh SG, Tackett AJ, Suva LJ, Eyre DR, Morello R (2016) Sc65-null mice provide evidence for a novel endoplasmic reticulum complex regulating collagen lysyl hydroxylation. PLoS Genet 12:e1006002

    Article  Google Scholar 

  5. Yamauchi M, Sricholpech M (2012) Lysine post-translational modifications of collagen. Essays Biochem 52:113–133

    Article  CAS  Google Scholar 

  6. Gjaltema RA, Bank RA (2017) Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease. Crit Rev Biochem Mol Biol 52:74–95

    Article  CAS  Google Scholar 

  7. Yamauchi M, Barker TH, Gibbons DL, Kurie JM (2018) The fibrotic tumor stroma. J Clin Invest 128:16–25

    Article  Google Scholar 

  8. Yamauchi M, Taga Y, Hattori S, Shiiba M, Terajima M (2018) Analysis of collagen and elastin cross-links. Methods Cell Biol 143:115–132

    Article  Google Scholar 

  9. Shinkai H, Yonemasu K (1979) Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens. Biochem J 177:847–852

    Article  CAS  Google Scholar 

  10. Miller EJ (1984) Collagen chemistry. In: Piez KA, Reddi AH (eds) Extracellular matrix biochemistry. Elsevier Science Publishing Co., Inc., Amsterdam, pp 41–78

    Google Scholar 

  11. Spiro RG (1967) The structure of the disaccharide unit of the renal glomerular basement membrane. J Biol Chem 242:4813–4823

    CAS  PubMed  Google Scholar 

  12. Kivirikko KI, Myllylä R (1982) Posttranslational enzymes in the biosynthesis of collagen: intracellular enzymes. Methods Enzymol 82:245–304

    Article  CAS  Google Scholar 

  13. Heikkinen J, Risteli M, Wang C, Latvala J, Rossi M, Valtavaara M, Myllyla R (2000) Lysyl hydroxylase 3 is a multifunctional protein possessing collagen glucosyltransferase activity. J Biol Chem 275:36158–36163

    Article  CAS  Google Scholar 

  14. Wang C, Luosujarvi H, Heikkinen J, Risteli M, Uitto L, Myllyla R (2002) The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro. Matrix Biol 21:559–566

    Article  CAS  Google Scholar 

  15. Rautavuoma K, Takaluoma K, Sormunen R, Myllyharju J, Kivirikko KI, Soininen R (2004) Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice. Proc Natl Acad Sci U S A 101:14120–14125

    Article  CAS  Google Scholar 

  16. Schegg B, Hulsmeier AJ, Rutschmann C, Maag C, Hennet T (2009) Core glycosylation of collagen is initiated by two beta(1-O)galactosyltransferases. Mol Cell Biol 29:943–952

    Article  CAS  Google Scholar 

  17. Ruotsalainen H, Sipila L, Vapola M, Sormunen R, Salo AM, Uitto L, Mercer DK, Robins SP, Risteli M, Aszodi A, Fassler R, Myllyla R (2006) Glycosylation catalyzed by lysyl hydroxylase 3 is essential for basement membranes. J Cell Sci 119:625–635

    Article  CAS  Google Scholar 

  18. Sipila L, Ruotsalainen H, Sormunen R, Baker NL, Lamande SR, Vapola M, Wang C, Sado Y, Aszodi A, Myllyla R (2007) Secretion and assembly of type IV and VI collagens depend on glycosylation of hydroxylysines. J Biol Chem 282:33381–33388

    Article  Google Scholar 

  19. Sricholpech M, Perdivara I, Nagaoka H, Yokoyama M, Tomer KB, Yamauchi M (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J Biol Chem 286:8846–8856

    Article  CAS  Google Scholar 

  20. Baumann S, Hennet T (2016) Collagen accumulation in osteosarcoma cells lacking GLT25D1 collagen galactosyltransferase. J Biol Chem 291:18514–18524

    Article  CAS  Google Scholar 

  21. Sricholpech M, Perdivara I, Yokoyama M, Nagaoka H, Terajima M, Tomer KB, Yamauchi M (2012) Lysyl Hydroxylase 3-mediated glucosylation in type i collagen: molecular loci and biological significance. J Biol Chem 287:22998–23009

    Article  CAS  Google Scholar 

  22. Morgan PH, Jacobs HG, Segrest JP, Cunningham LW (1970) A comparative study of glycopeptides derived from selected vertebrate collagens. A possible role of the carbohydrate in fibril formation. J Biol Chem 245:5042–5048

    CAS  PubMed  Google Scholar 

  23. Aguilar JH, Jacobs HG, Butler WT, Cunningham LW (1973) The distribution of carbohydrate groups in rat skin collagen. J Biol Chem 248:5106–5013

    CAS  PubMed  Google Scholar 

  24. Fietzek PP, Kuhn K (1976) The primary structure of collagen. Int Rev Connect Tissue Res 7:1–60

    Article  CAS  Google Scholar 

  25. Schofield JD, Freeman IL, Jackson DS (1971) The isolation, and amino acid and carbohydrate composition, of polymeric collagens prepared from various human tissues. Biochem J 124:467–473

    Article  CAS  Google Scholar 

  26. Toole BP, Kang AH, Trelstad RL, Gross J (1972) Collagen heterogeneity within different growth regions of long bones of rachitic and non-rachitic chicks. Biochem J 127:715–720

    Article  CAS  Google Scholar 

  27. Michalsky M, Norris-Suarez K, Bettica P, Pecile A, Moro L (1993) Rat cortical and trabecular bone collagen glycosylation are differently influenced by ovariectomy. Biochem Biophys Res Commun 192:1281–1288

    Article  CAS  Google Scholar 

  28. Tenni R, Valli M, Rossi A, Cetta G (1993) Possible role of overglycosylation in the type I collagen triple helical domain in the molecular pathogenesis of osteogenesis imperfecta. Am J Med Genet 45:252–256

    Article  CAS  Google Scholar 

  29. Lehmann HW, Wolf E, Roser K, Bodo M, Delling G, Muller PK (1995) Composition and posttranslational modification of individual collagen chains from osteosarcomas and osteofibrous dysplasias. J Cancer Res Clin Oncol 121:413–418

    Article  CAS  Google Scholar 

  30. Brinckmann J, Notbohm H, Tronnier M, Acil Y, Fietzek PP, Schmeller W, Muller PK, Batge B (1999) Overhydroxylation of lysyl residues is the initial step for altered collagen cross-links and fibril architecture in fibrotic skin. J Invest Dermatol 113:617–621

    Article  CAS  Google Scholar 

  31. Salo AM, Cox H, Farndon P, Moss C, Grindulis H, Risteli M, Robins SP, Myllyla R (2008) A connective tissue disorder caused by mutations of the lysyl hydroxylase 3 gene. Am J Hum Genet 83:495–503

    Article  CAS  Google Scholar 

  32. Risteli M, Ruotsalainen H, Salo AM, Sormunen R, Sipila L, Baker NL, Lamande SR, Vimpari-Kauppinen L, Myllyla R (2009) Reduction of lysyl hydroxylase 3 causes deleterious changes in the deposition and organization of extracellular matrix. J Biol Chem 284:28204–28211

    Article  CAS  Google Scholar 

  33. Brenner RE, Vetter U, Nerlich A, Worsdorfer O, Teller WM, Muller PK (1990) Altered collagen metabolism in osteogenesis imperfecta fibroblasts: a study on 33 patients with diverse forms. Eur J Clin Investig 20:8–14

    Article  CAS  Google Scholar 

  34. Bateman JF, Mascara T, Chan D, Cole WG (1984) Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta. Biochem J 217:103–115

    Article  CAS  Google Scholar 

  35. Cetta G, De Luca G, Tenni R, Zanaboni G, Lenzi L, Castellani AA (1983) Biochemical investigations of different forms of osteogenesis imperfecta. Evaluation of 44 cases. Connect Tissue Res 11:103–111

    Article  CAS  Google Scholar 

  36. Moro L, Bettica P, Romanello M, Suarez KN (1997) 17 beta-Estradiol and tamoxifen prevent the over-glycosylation of rat trabecular bone collagen induced by ovariectomy. Eur J Clin Chem Clin Biochem 35:29–33

    CAS  PubMed  Google Scholar 

  37. Yang C, Niu C, Bodo M, Gabriel E, Notbohm H, Wolf E, Muller PK (1993) Fulvic acid supplementation and selenium deficiency disturb the structural integrity of mouse skeletal tissue. An animal model to study the molecular defects of Kashin-Beck disease. Biochem J 289(Pt 3):829–835

    Article  CAS  Google Scholar 

  38. Amudeswari S, Liang JN, Chakrabarti B (1987) Polar-apolar characteristics and fibrillogenesis of glycosylated collagen. Coll Relat Res 7:215–223

    Article  CAS  Google Scholar 

  39. Torre-Blanco A, Adachi E, Hojima Y, Wootton JA, Minor RR, Prockop DJ (1992) Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils. J Biol Chem 267:2650–2655

    CAS  PubMed  Google Scholar 

  40. Yang CL, Rui H, Mosler S, Notbohm H, Sawaryn A, Muller PK (1993) Collagen II from articular cartilage and annulus fibrosus. Structural and functional implication of tissue specific posttranslational modifications of collagen molecules. Eur J Biochem 213:1297–1302

    Article  CAS  Google Scholar 

  41. Notbohm H, Nokelainen M, Myllyharju J, Fietzek PP, Muller PK, Kivirikko KI (1999) Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro. J Biol Chem 274:8988–8992

    Article  CAS  Google Scholar 

  42. Batge B, Winter C, Notbohm H, Acil Y, Brinckmann J, Muller PK (1997) Glycosylation of human bone collagen I in relation to lysylhydroxylation and fibril diameter. J Biochem 122:109–115

    Article  CAS  Google Scholar 

  43. Eyre DR, Glimcher MJ (1973) Analysis of a cross-linked peptide from calf bone collagen: evidence that hydroxylysyl glycoside participates in the crosslink. Biochem Biophys Res Commun 52:663–671

    Article  CAS  Google Scholar 

  44. Hanson DA, Eyre DR (1996) Molecular site specificity of pyridinoline and pyrrole cross-links in type i collagen of human bone. J Biol Chem 271:26508–26516

    Article  CAS  Google Scholar 

  45. Robins SP (1983) Cross-linking of collagen. Isolation, structural characterization and glycosylation of pyridinoline. Biochem J 215:167–173

    Article  CAS  Google Scholar 

  46. Yamauchi M, Noyes C, Kuboki Y, Mechanic GL (1982) Collagen structural microheterogeneity and a possible role for glycosylated hydroxylysine in type I collagen. Proc Natl Acad Sci U S A 79:7684–7688

    Article  CAS  Google Scholar 

  47. Yamauchi M, Katz EP, Mechanic GL (1986) Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament. Biochemistry 25:4907–4913

    Article  CAS  Google Scholar 

  48. Vogel W, Gish GD, Alves F, Pawson T (1997) The discoidin domain receptor tyrosine kinases are activated by collagen. Mol Cell 1:13–23

    Article  CAS  Google Scholar 

  49. Jurgensen HJ, Madsen DH, Ingvarsen S, Melander MC, Gardsvoll H, Patthy L, Engelholm LH, Behrendt N (2011) A novel functional role of collagen glycosylation: interaction with the endocytic collagen receptor uparap/ENDO180. J Biol Chem 286:32736–32748

    Article  Google Scholar 

  50. Palmieri D, Valli M, Viglio S, Ferrari N, Ledda B, Volta C, Manduca P (2010) Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I. Exp Cell Res 316:789–799

    Article  CAS  Google Scholar 

  51. Butler WT, Cunningham LW (1966) Evidence for the linkage of a disaccharide to hydroxylysine in tropocollagen. J Biol Chem 241:3882–3888

    CAS  PubMed  Google Scholar 

  52. Perdivara I, Perera L, Sricholpech M, Terajima M, Pleshko N, Yamauchi M, Tomer KB (2013) Unusual fragmentation pathways in collagen glycopeptides. J Am Soc Mass Spectrom 24:1072–1081

    Article  CAS  Google Scholar 

  53. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S (2012) Development of a novel method for analyzing collagen O-glycosylations by hydrazide chemistry. Mol Cell Proteomics 11:M111 010397

    Article  Google Scholar 

  54. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S (2013) Site-specific quantitative analysis of overglycosylation of collagen in osteogenesis imperfecta using hydrazide chemistry and SILAC. J Proteome Res 12:2225–2232

    Article  CAS  Google Scholar 

  55. Pornprasertsuk S, Duarte WR, Mochida Y, Yamauchi M (2004) Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells. J Bone Miner Res 19:1349–1355

    Article  CAS  Google Scholar 

  56. Yamauchi M, Shiiba M (2008) Lysine hydroxylation and cross-linking of collagen. Methods Mol Biol 446:95–108

    Article  CAS  Google Scholar 

  57. Eyre D (1987) Collagen cross-linking amino acids. In: Colowick SP, Kaplan NO (eds) Methods in enzymology. Academic Press, Inc., Orlando, pp 115–139

    Google Scholar 

  58. Perdivara I, Yamauchi M, Tomer KB (2013) Molecular characterization of collagen hydroxylysine O-glycosylation by mass spectrometry: current status. Aust J Chem 66:760–769

    Article  CAS  Google Scholar 

  59. Terajima M, Perdivara I, Sricholpech M, Deguchi Y, Pleshko N, Tomer KB, Yamauchi M (2014) Glycosylation and cross-linking in bone type I collagen. J Biol Chem 289:22636–22647

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by National Institutes of Health Grants DE020909 and AR060978 (to M.Y.).

Author information

Authors and Affiliations

  1. Department of Oral and Craniofacial Health Sciences, School of Dentistry, University of North Carolina, Chapel Hill, NC, USA

    Mitsuo Yamauchi & Masahiko Terajima

  2. Faculty of Dentistry, Department of Oral Surgery and Oral Medicine, Srinakharinwirot University, Bangkok, Thailand

    Marnisa Sricholpech

  3. Cary, NC, USA

    Kenneth B. Tomer

  4. Fujifilm Diosynth Biotechnologies, Morrisville, NC, USA

    Irina Perdivara

Authors
  1. Mitsuo Yamauchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marnisa Sricholpech
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masahiko Terajima
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kenneth B. Tomer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Irina Perdivara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mitsuo Yamauchi .

Editor information

Editors and Affiliations

  1. Recombinant Research and Development, Octapharma Biopharmaceuticals GmbH, Heidelberg, Germany

    Christoph Kannicht

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Yamauchi, M., Sricholpech, M., Terajima, M., Tomer, K.B., Perdivara, I. (2019). Glycosylation of Type I Collagen. In: Kannicht, C. (eds) Post-Translational Modification of Proteins. Methods in Molecular Biology, vol 1934. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9055-9_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9055-9_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9053-5

  • Online ISBN: 978-1-4939-9055-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation