Skip to main content
SpringerLink
Log in

The Detection of Glycosaminoglycans in Pancreatic Islets and Lymphoid Tissues

  • Protocol
  • First Online:
Glycosaminoglycans

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1229))

Abstract

In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage, and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP–link protein complex is applied to tissue sections and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates.

To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP. The hyaluronan–HABP complexes are extracted and the hyaluronan concentration in the tissue is determined using an ELISA-like assay.

Heparan sulfate is identified in mouse tissues by Alcian blue histochemistry and indirect immunohistochemistry. In human tissues, heparan sulfate is best detected by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A biotinylated secondary antibody is then applied in conjunction with streptavidin-peroxidase and its binding to the anti-heparan sulfate antibody is visualized by enzymatic precipitation of chromogenic substrates.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Laurent TC, Laurent UB, Fraser JR (1996) The structure and function of hyaluronan: an overview. Immunol Cell Biol 74:A1–A7

    Article  CAS  PubMed  Google Scholar 

  2. Stern R, Asari AA, Sugahara KN (2006) Hyaluronan fragments: an information-rich system. Eur J Cell Biol 85:699–715

    Article  CAS  PubMed  Google Scholar 

  3. Jiang D, Liang J, Noble PW (2011) Hyaluronan as an immune regulator in human diseases. Physiol Rev 91:221–264

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Perrimon N, Bernfield M (2000) Specificities of heparan sulphate proteoglycans in developmental processes. Nature 404:725–728

    Article  CAS  PubMed  Google Scholar 

  5. Iozzo RV (2001) Heparan sulfate proteoglycans: intricate molecules with intriguing functions. J Clin Invest 108:165–167

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Parish CR (2006) The role of heparan sulphate in inflammation. Nat Rev Immunol 6:633–643

    Article  CAS  PubMed  Google Scholar 

  7. Hull RL, Johnson PY, Braun KR, Day AJ, Wight TN (2012) Hyaluronan and hyaluronan binding proteins are normal components of mouse pancreatic islets and are differentially expressed by islet endocrine cell types. J Histochem Cytochem 60:749–760

    Article  PubMed Central  PubMed  Google Scholar 

  8. Bogdani M, Johnson PY, Potter-Perigo S, Nagy N, Day AJ, Bollyky PL, Wight TN (2014) Hyaluronan and hyaluronan binding proteins accumulate in both human type 1 diabetic islets and lymphoid tissues and associate with inflammatory cells in insulitis. Diabetes 63:2727–2743

    Google Scholar 

  9. Irving-Rodgers HF, Ziolkowski AF, Parish CR, Sado Y, Ninomiya Y, Simeonovic CJ, Rodgers RJ (2008) Molecular composition of the peri-islet basement membrane in NOD mice: a barrier against destructive insulitis. Diabetologia 51:1680–1688

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Ziolkowski AF, Popp SK, Freeman C, Parish CR, Simeonovic CJ (2012) Heparan sulfate and heparanase play key roles in mouse β cell survival and autoimmune diabetes. J Clin Invest 122:132–141

    Google Scholar 

  11. Brown TJ, Kimpton WG, Fraser JR (2000) Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node. Glycoconj J 17:795–805

    Article  CAS  PubMed  Google Scholar 

  12. Kramer RH, Rosen SD, McDonald KA (1988) Basement-membrane components associated with the extracellular matrix of the lymph node. Cell Tissue Res 252:367–375

    Article  CAS  PubMed  Google Scholar 

  13. Kaldjian EP, Gretz JE, Anderson AO, Shi Y, Shaw S (2001) Spatial and molecular organization of lymph node T cell cortex: a labyrinthine cavity bounded by an epithelium-like monolayer of fibroblastic reticular cells anchored to basement membrane-like extracellular matrix. Int Immunol 13:1243–1253

    Article  CAS  PubMed  Google Scholar 

  14. Tengblad A (1979) Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding proteins from cartilage. Biochim Biophys Acta 578:281–289

    Article  CAS  PubMed  Google Scholar 

  15. Ripellino JA, Klinger MM, Margolis RU, Margolis RK (1985) The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. J Histochem Cytochem 33:1060–1066

    Article  CAS  PubMed  Google Scholar 

  16. Knudson CB, Toole BP (1985) Fluorescent morphological probe for hyaluronate. J Cell Biol 100:1753–1758

    Article  CAS  PubMed  Google Scholar 

  17. Ripellino JA, Bailo M, Margolis RU, Margolis RK (1988) Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum. J Cell Biol 106:845–855

    Article  CAS  PubMed  Google Scholar 

  18. Banerjee SD, Toole BP (1991) Monoclonal antibody to chick embryo hyaluronan-binding protein: changes in distribution of binding protein during early brain development. Dev Biol 146:186–197

    Article  CAS  PubMed  Google Scholar 

  19. Azumi N, Underhill CB, Kagan E, Sheibani K (1992) A novel biotinylated probe specific for hyaluronate. Its diagnostic value in diffuse malignant mesothelioma. Am J Surg Pathol 16:116–121

    Article  CAS  PubMed  Google Scholar 

  20. Underhill CB, Nguyen HA, Shizari M, Culty M (1993) CD44 positive macrophages take up hyaluronan during lung development. Dev Biol 155:324–336

    Article  CAS  PubMed  Google Scholar 

  21. Toole BP, Yu Q, Underhill CB (2001) Hyaluronan and hyaluronan-binding proteins. Probes for specific detection. Methods Mol Biol 171:479–485

    CAS  PubMed  Google Scholar 

  22. Toole BP (2004) Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 4:528–539

    Article  CAS  PubMed  Google Scholar 

  23. Underhill CB, Zhang L (2000) Analysis of hyaluronan using biotinylated hyaluronan-binding proteins. Methods Mol Biol 137:441–447

    CAS  PubMed  Google Scholar 

  24. Campbell-Thompson ML, Montgomery EL, Foss RM, Kolheffer KM, Phipps G, Schneider L, Atkinson MA (2012) Collection protocol for human pancreas. J Vis Exp 63:e4039

    PubMed  Google Scholar 

  25. Campbell-Thompson M, Wasserfall C, Kaddis J, Albanese-O'Neill A, Staeva T, Nierras C, Moraski J, Rowe P, Gianani R, Eisenbarth G, Crawford J, Schatz D, Pugliese A, Atkinson M (2012) Network for Pancreatic Organ Donors with Diabetes (nPOD): developing a tissue biobank for type 1 diabetes. Diabetes Metab Res Rev 28:608–617

    Article  PubMed Central  PubMed  Google Scholar 

  26. AVMA (American Veterinary Medical Association), Guidelines for the Euthanasia of Animals: 2013 Edition, Schaumburg, IL

    Google Scholar 

  27. Artwohl J, Brown P, Corning B, Stein S (2006) Report of the ACLAM Task Force on rodent euthanasia. J Am Assoc Lab Anim Sci 45:98–105

    CAS  PubMed  Google Scholar 

  28. Haserodt S, Aytekin M, Dweik RA (2011) A comparison of the sensitivity, specificity, and molecular weight accuracy of three different commercially available Hyaluronan ELISA-like assays. Glycobiology 21:175–183

    Article  CAS  PubMed  Google Scholar 

  29. Scott JE, Dorling J (1965) Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie 5:221–233

    Article  CAS  PubMed  Google Scholar 

  30. Calvitti M, Baroni T, Calastrini C, Lilli C, Caramelli E, Becchetti E, Carinci P, Vizzotto L, Stabellini G (2004) Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development. J Histochem Cytochem 52:325–334

    Article  CAS  PubMed  Google Scholar 

  31. Yingsung W, Zhuo L, Morgelin M, Yoneda M, Kida D, Watanabe H, Ishiguro N, Iwata H, Kimata K (2003) Molecular heterogeneity of the SHAP-hyaluronan complex. Isolation and characterization of the complex in synovial fluid from patients with rheumatoid arthritis. J Biol Chem 278:32710–32718

    Article  CAS  PubMed  Google Scholar 

  32. David G, Bai X, Van der Schueren B, Cassiman JJ, Van den Berghe H (1992) Developmental changes in heparan sulfate expression: in situ detection with monoclonal antibodies. J Cell Biol 119:961–975

    Article  CAS  PubMed  Google Scholar 

  33. van den Born J, Salmivirta K, Henttinen T, Ostman N, Ishimaru T, Miyaura S, Yoshida K, Salmivirta M (2005) Novel heparan sulfate structures revealed by monoclonal antibodies. J Biol Chem 280:20516–20523

    Article  PubMed  Google Scholar 

  34. Tammi R, Ripellino JA, Margolis RU, Tammi M (1988) Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J Invest Dermatol 90:412–414

    Article  CAS  PubMed  Google Scholar 

  35. Tammi R, Tammi M, Hakkinen L, Larjava H (1990) Histochemical localization of hyaluronate in human oral epithelium using a specific hyaluronate-binding probe. Arch Oral Biol 35:219–224

    Article  CAS  PubMed  Google Scholar 

  36. Parkkinen JJ, Hakkinen TP, Savolainen S, Wang C, Tammi R, Agren UM, Lammi MJ, Arokoski J, Helminen HJ, Tammi MI (1996) Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan. Histochem Cell Biol 105:187–194

    Article  CAS  PubMed  Google Scholar 

  37. Wang C, Tammi M, Guo H, Tammi R (1996) Hyaluronan distribution in the normal epithelium of esophagus, stomach, and colon and their cancers. Am J Pathol 148:1861–1869

    CAS  PubMed Central  PubMed  Google Scholar 

  38. Anttila MA, Tammi RH, Tammi MI, Syrjanen KJ, Saarikoski SV, Kosma VM (2000) High levels of stromal hyaluronan predict poor disease outcome in epithelial ovarian cancer. Cancer Res 60:150–155

    CAS  PubMed  Google Scholar 

  39. Bohm J, Niskanen L, Tammi R, Tammi M, Eskelinen M, Pirinen R, Hollmen S, Alhava E, Kosma VM (2002) Hyaluronan expression in differentiated thyroid carcinoma. J Pathol 196:180–185

    Article  CAS  PubMed  Google Scholar 

  40. Auvinen P, Tammi R, Kosma VM, Sironen R, Soini Y, Mannermaa A, Tumelius R, Uljas E, Tammi M (2013) Increased hyaluronan content and stromal cell CD44 associate with HER2 positivity and poor prognosis in human breast cancer. Int J Cancer 132:531–539

    Article  CAS  PubMed  Google Scholar 

  41. de la Motte CA, Hascall VC, Drazba J, Bandyopadhyay SK, Strong SA (2003) Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-a-trypsin inhibitor is crucial to structure and function. Am J Pathol 163:121–133

    Article  PubMed Central  PubMed  Google Scholar 

  42. Selbi W, de la Motte CA, Hascall VC, Day AJ, Bowen T, Phillips AO (2006) Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells. Kidney Int 70:1287–1295

    Article  CAS  PubMed  Google Scholar 

  43. Aytekin M, Comhair SA, de la Motte C, Bandyopadhyay SK, Farver CF, Hascall VC, Erzurum SC, Dweik RA (2008) High levels of hyaluronan in idiopathic pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 295:L789–L799

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Lewis A, Steadman R, Manley P, Craig K, de la Motte C, Hascall V, Phillips AO (2008) Diabetic nephropathy, inflammation, hyaluronan and interstitial fibrosis. Histol Histopathol 23:731–739

    PubMed  Google Scholar 

  45. de la Motte CA, Drazba JA (2011) Viewing hyaluronan: imaging contributes to imagining new roles for this amazing matrix polymer. J Histochem Cytochem 59:252–257

    Article  PubMed Central  PubMed  Google Scholar 

  46. Boregowda RK, Appaiah HN, Siddaiah M, Kumarswamy SB, Sunila S, Thimmaiah KN, Mortha K, Toole B, Banerjee S (2006) Expression of hyaluronan in human tumor progression. J Carcinog 5:2

    Article  PubMed Central  PubMed  Google Scholar 

  47. Tan KT, McGrouther DA, Day AJ, Milner CM, Bayat A (2011) Characterization of hyaluronan and TSG-6 in skin scarring: differential distribution in keloid scars, normal scars and unscarred skin. J Eur Acad Dermatol Venereol 25:317–327

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Lin W, Shuster S, Maibach HI, Stern R (1997) Patterns of hyaluronan staining are modified by fixation techniques. J Histochem Cytochem 45:1157–1163

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was performed with the support of the Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative type 1 diabetes research project sponsored by JDRF, Grant 25-2010-648, National Institutes of Health grants U01 AI101984, CSGADP Innovative Project (under AI101984), and P01 HL098067 (T.N.W.). Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at www.jdrfnpod.org/our-partners.php. This work was also supported by a National Health and Medical Research Council of Australia (NH&MRC)/Juvenile Diabetes Research Foundation (JDRF) Special Program Grant in Type 1 Diabetes (#418138; C.S.), a NHMRC Project Grant (#1043284), JDRF nPOD Research Grant 25-2010-716 (C.S.), a research grant from the Roche Organ Transplantation Research Foundation (ROTRF)/JDRF (#477554991; C.S.), and a Deutsche Forschungsgemeinschaft (DFG) Research Grant NA 965/2-1 (N.N.). We thank Anne Prins for assistance with the Alcian blue histochemical methodology and Lora Jensen and Sarah Popp for optimizing the heparan sulfate immunohistochemistry on nPOD human pancreas sections.

Author information

Authors and Affiliations

  1. Matrix Biology Program, Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle, WA, 98101, USA

    Marika Bogdani, Nadine Nagy, Pamela Y. Johnson, Christina K. Chan & Thomas N. Wight Ph.D.

  2. Diabetes/Transplantation Immunobiology Laboratory, Department of Immunology, The John Curtin School of Medical Research, The Australian National University, GPO Box 334, Canberra, ACT, 2601, Australia

    Charmaine Simeonovic

Authors
  1. Marika Bogdani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Charmaine Simeonovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nadine Nagy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pamela Y. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christina K. Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas N. Wight Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas N. Wight Ph.D. .

Editor information

Editors and Affiliations

  1. Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USA

    Kuberan Balagurunathan

  2. Department of Genetics, Cell Biology and Development, The University of Minnesota, Minneapolis, Minnesota, USA

    Hiroshi Nakato

  3. Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia, USA

    Umesh R. Desai

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Bogdani, M., Simeonovic, C., Nagy, N., Johnson, P.Y., Chan, C.K., Wight, T.N. (2015). The Detection of Glycosaminoglycans in Pancreatic Islets and Lymphoid Tissues. In: Balagurunathan, K., Nakato, H., Desai, U. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 1229. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1714-3_32

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1714-3_32

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1713-6

  • Online ISBN: 978-1-4939-1714-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation