Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Am J Pathol. 1989 Oct; 135(4): 637–646.
PMCID: PMC1880029
PMID: 2529772

Basement membrane heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture.

J. L. Stow, C. J. Soroka, K. MacKay, L. Striker, G. Striker, and M. G. Farquhar
Author information Copyright and License information PMC Disclaimer

Abstract

The production and distribution of basement membrane-type heparan sulfate proteoglycans (BM HSPG) were investigated in a mouse glomerular epithelial cell line. Confluent cell monolayers were radiolabeled with [35S]sulfate or [35S]cysteine. Proteoglycans were isolated from the medium and cell layers by ion exchange chromatography and their nature determined by enzyme digestion (chondroitinase ABC) or degradative treatment (nitrous acid). It was found that more than 80% of the proteoglycans in both the cell layer and medium were heparan sulfate proteoglycans (HSPG) based on their susceptibility to nitrous acid degradation. More than half of the HSPG in the cell layer could be precipitated with an antiserum that specifically recognizes BM HSPG; only 10% of those released into the medium were precipitated with this antiserum. When immunoprecipitates of [35S] sulfate-labeled proteoglycans were analyzed by SDS-PAGE, the mature proteoglycans ran as a broad band at the top of the gel. When immunoprecipitates of [35S]cysteine-labeled proteoglycans were similarly analyzed, a 250 kd precursor core protein band was seen in addition to the mature proteoglycan. When BM HSPG were localized by immunofluorescence and immunoelectron microscopy (immunoperoxidase), they were found intracellularly in biosynthetic compartments (ER and Golgi cisternae) and extracellularly in deposits of basement membrane-like matrix located beneath and between the cells. These results indicate that l) BM HSPG are the predominant type of proteoglycans made by glomerular epithelial cells in culture; 2) these HSPG are assembled into a loosely organized matrix that is deposited beneath and between the cells; and 3) this cell type produces a higher proportion of BM HSPG than other cultured epithelial cells studied previously.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

 
icon of scanned page 637
637
icon of scanned page 638
638
icon of scanned page 639
639
icon of scanned page 640
640
icon of scanned page 641
641
icon of scanned page 642
642
icon of scanned page 643
643
icon of scanned page 644
644
icon of scanned page 645
645
icon of scanned page 646
646
 

Images in this article

Figure 6

Figure 6
on p.644

Figure 1

Figure 1
on p.639

Figure 5

Figure 5
on p.643

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  • Kanwar YS. Biophysiology of glomerular filtration and proteinuria. Lab Invest. 1984 Jul;51(1):7–21. [PubMed] [Google Scholar]
  • Shimomura H, Spiro RG. Studies on macromolecular components of human glomerular basement membrane and alterations in diabetes. Decreased levels of heparan sulfate proteoglycan and laminin. Diabetes. 1987 Mar;36(3):374–381. [PubMed] [Google Scholar]
  • Cohen MP, Surma ML. [(35)S]sulfate incorporation into glomerular basement membrane glycosaminoglycans is decreased in experimental diabetes. J Lab Clin Med. 1981 Nov;98(5):715–722. [PubMed] [Google Scholar]
  • Kanwar YS, Rosenzweig LJ, Linker A, Jakubowski ML. Decreased de novo synthesis of glomerular proteoglycans in diabetes: biochemical and autoradiographic evidence. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2272–2275. [PMC free article] [PubMed] [Google Scholar]
  • Rohrbach DH, Wagner CW, Star VL, Martin GR, Brown KS, Yoon JW. Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice. J Biol Chem. 1983 Oct 10;258(19):11672–11677. [PubMed] [Google Scholar]
  • Caulfield JP, Farquhar MG. Loss of anionic sites from the glomerular basement membrane in aminonucleoside nephrosis. Lab Invest. 1978 Nov;39(5):505–512. [PubMed] [Google Scholar]
  • Klein DJ, Dehnel PJ, Oegema TR, Brown DM. Alterations in proteoglycan metabolism in the nephrotic syndrome induced by the aminonucleoside of puromycin. Lab Invest. 1984 May;50(5):543–551. [PubMed] [Google Scholar]
  • Mynderse LA, Hassell JR, Kleinman HK, Martin GR, Martinez-Hernandez A. Loss of heparan sulfate proteoglycan from glomerular basement membrane of nephrotic rats. Lab Invest. 1983 Mar;48(3):292–302. [PubMed] [Google Scholar]
  • Vernier RL, Klein DJ, Sisson SP, Mahan JD, Oegema TR, Brown DM. Heparan sulfate--rich anionic sites in the human glomerular basement membrane. Decreased concentration in congenital nephrotic syndrome. N Engl J Med. 1983 Oct 27;309(17):1001–1009. [PubMed] [Google Scholar]
  • Schneeberger EE, Stavrakis G, McCarthy K. Alterations in glomerular anionic sites in autologous immune complex nephritis. Lab Invest. 1983 Oct;49(4):445–452. [PubMed] [Google Scholar]
  • Melnick GF, Ladoulis CT, Cavallo T. Decreased anionic groups and increased permeability precedes deposition of immune complexes in the glomerular capillary wall. Am J Pathol. 1981 Nov;105(2):114–120. [PMC free article] [PubMed] [Google Scholar]
  • Stow JL, Sawada H, Farquhar MG. Basement membrane heparan sulfate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus. Proc Natl Acad Sci U S A. 1985 May;82(10):3296–3300. [PMC free article] [PubMed] [Google Scholar]
  • Striker GE, Killen PD, Farin FM. Human glomerular cells in vitro: isolation and characterization. Transplant Proc. 1980 Sep;12(3 Suppl 1):88–99. [PubMed] [Google Scholar]
  • Foidart JM, Foidart JB, Mahieu PR. Synthesis of collagen and fibronectin by glomerular cells in culture. Ren Physiol. 1980;3(1-6):183–192. [PubMed] [Google Scholar]
  • Striker GE, Striker LJ. Glomerular cell culture. Lab Invest. 1985 Aug;53(2):122–131. [PubMed] [Google Scholar]
  • MacKay K, Striker LJ, Elliot S, Pinkert CA, Brinster RL, Striker GE. Glomerular epithelial, mesangial, and endothelial cell lines from transgenic mice. Kidney Int. 1988 Mar;33(3):677–684. [PubMed] [Google Scholar]
  • Stow JL, Kjéllen L, Unger E, Hök M, Farquhar MG. Heparan sulfate proteoglycans are concentrated on the sinusoidal plasmalemmal domain and in intracellular organelles of hepatocytes. J Cell Biol. 1985 Mar;100(3):975–980. [PMC free article] [PubMed] [Google Scholar]
  • Stow JL, Farquhar MG. Distinctive populations of basement membrane and cell membrane heparan sulfate proteoglycans are produced by cultured cell lines. J Cell Biol. 1987 Jul;105(1):529–539. [PMC free article] [PubMed] [Google Scholar]
  • Cifonelli JA, King J. The distribution of 2-acetamido-2-deoxy-D-glucose residues in mammalian heparins. Carbohydr Res. 1972 Feb;21(2):173–186. [PubMed] [Google Scholar]
  • Carlson DM. Structures and immunochemical properties of oligosaccharides isolated from pig submaxillary mucins. J Biol Chem. 1968 Feb 10;243(3):616–626. [PubMed] [Google Scholar]
  • Kreisberg JI, Karnovsky MJ. Glomerular cells in culture. Kidney Int. 1983 Mar;23(3):439–447. [PubMed] [Google Scholar]
  • Oohira A, Wight TN, Bornstein P. Sulfated proteoglycans synthesized by vascular endothelial cells in culture. J Biol Chem. 1983 Feb 10;258(3):2014–2021. [PubMed] [Google Scholar]
  • Kanwar YS, Veis A, Kimura JH, Jakubowski ML. Characterization of heparan sulfate-proteoglycan of glomerular basement membranes. Proc Natl Acad Sci U S A. 1984 Feb;81(3):762–766. [PMC free article] [PubMed] [Google Scholar]
  • Kato M, Koike Y, Ito Y, Suzuki S, Kimata K. Multiple forms of heparan sulfate proteoglycans in the Engelbreth-Holm-Swarm mouse tumor. The occurrence of high density forms bearing both heparan sulfate and chondroitin sulfate side chains. J Biol Chem. 1987 May 25;262(15):7180–7188. [PubMed] [Google Scholar]
  • Farquhar MG. Progress in unraveling pathways of Golgi traffic. Annu Rev Cell Biol. 1985;1:447–488. [PubMed] [Google Scholar]
  • Lemkin MC, Farquhar MG. Sulfated and nonsulfated glycosaminoglycans and glycopeptides are synthesized by kidney in vivo and incorporated into glomerular basement membranes. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1726–1730. [PMC free article] [PubMed] [Google Scholar]
  • Kanwar YS, Hascall VC, Farquhar MG. Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane. J Cell Biol. 1981 Aug;90(2):527–532. [PMC free article] [PubMed] [Google Scholar]
  • Klein DJ, Brown DM, Oegema TR, Brenchley PE, Anderson JC, Dickinson MA, Horigan EA, Hassell JR. Glomerular basement membrane proteoglycans are derived from a large precursor. J Cell Biol. 1988 Mar;106(3):963–970. [PMC free article] [PubMed] [Google Scholar]
  • Stow JL, Glasgow EF, Handley CJ, Hascall VC. Biosynthesis of proteoglycans by isolated rabbit glomeruli. Arch Biochem Biophys. 1983 Sep;225(2):950–957. [PubMed] [Google Scholar]
  • Klein DJ, Brown DM, Oegema TR., Jr Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli. J Biol Chem. 1986 Dec 15;261(35):16636–16652. [PubMed] [Google Scholar]
  • Hök M, Kjellén L, Johansson S. Cell-surface glycosaminoglycans. Annu Rev Biochem. 1984;53:847–869. [PubMed] [Google Scholar]
  • Lark MW, Culp LA. Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. Affinity fractionation on columns of platelet factor 4, plasma fibronectin, and octyl-sepharose. J Biol Chem. 1984 Jun 10;259(11):6773–6782. [PubMed] [Google Scholar]
  • Rapraeger A, Jalkanen M, Bernfield M. Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells. J Cell Biol. 1986 Dec;103(6 Pt 2):2683–2696. [PMC free article] [PubMed] [Google Scholar]
  • Carey DJ, Todd MS. A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells. J Biol Chem. 1986 Jun 5;261(16):7518–7525. [PubMed] [Google Scholar]
  • Woods A, Hök M, Kjellén L, Smith CG, Rees DA. Relationship of heparan sulfate proteoglycans to the cytoskeleton and extracellular matrix of cultured fibroblasts. J Cell Biol. 1984 Nov;99(5):1743–1753. [PMC free article] [PubMed] [Google Scholar]
  • Bienkowski MJ, Conrad HE. Kinetics of proteoheparan sulfate synthesis, secretion, endocytosis, and catabolism by a hepatocyte cell line. J Biol Chem. 1984 Nov 10;259(21):12989–12996. [PubMed] [Google Scholar]
  • Brauker JH, Wang JL. Nonlysosomal processing of cell-surface heparan sulfate proteoglycans. Studies of I-cells and NH4Cl-treated normal cells. J Biol Chem. 1987 Sep 25;262(27):13093–13101. [PubMed] [Google Scholar]
  • Yanagishita M, Hascall VC. Metabolism of proteoglycans in rat ovarian granulosa cell culture. Multiple intracellular degradative pathways and the effect of chloroquine. J Biol Chem. 1984 Aug 25;259(16):10270–10283. [PubMed] [Google Scholar]
  • Piepkorn M, Hovingh P, Linker A. Evidence for independent metabolism and cell surface localization of cell surface localization of cellular proteoglycans and glycosaminoglycan free chains. J Cell Physiol. 1988 May;135(2):189–199. [PubMed] [Google Scholar]
  • Kanwar YS, Rosenzweig LJ, Jakubowski ML. Distribution of de novo synthesized sulfated glycosaminoglycans in the glomerular basement membrane and mesangial matrix. Lab Invest. 1983 Aug;49(2):216–225. [PubMed] [Google Scholar]
  • Hayman EG, Oldberg A, Martin GR, Ruoslahti E. Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells. J Cell Biol. 1982 Jul;94(1):28–35. [PMC free article] [PubMed] [Google Scholar]
Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology
-