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• W2005294501 abstract "The present study shows that collagen XVIII is, next to perlecan and agrin, the third basal lamina heparan sulfate proteoglycan (HSPG) and the first collagen/proteoglycan with heparan sulfate side chains. By using monoclonal antibodies to an unidentified HSPG in chick, 14 cDNA clones were isolated from a chick yolk sac library. All clones had a common nucleotide sequence that was homologous to the mRNA sequences of mouse and human collagen XVIII. The deduced amino acid sequence of the chick fragment shows an 83% overall homology with the human and mouse collagen XVIII. Similar to the human and mouse homologue, the chick collagen XVIII mRNA has a size of 4.5 kilobase pairs. In Western blots, collagen XVIII appeared as a smear with a molecular mass of 300 kDa. After treatment with heparitinase, the protein was reduced in molecular mass by 120 kDa to a protein core of 180 kDa. Collagen XVIII has typical features of a collagen, such as its existence, under non-denaturing conditions, as a non-covalently linked oligomer, and a sensitivity of the core protein to collagenase digestion. It also has characteristics of an HSPG, such as long heparitinase-sensitive carbohydrate chains and a highly negative net charge. Collagen XVIII is abundant in basal laminae of the retina, epidermis, pia, cardiac and striated muscle, kidney, blood vessels, and lung. In situ hybridization showed that the main expression of collagen XVIII HSPG in the chick embryo is in the kidney and the peripheral nervous system. As a substrate, collagen XVIII moderately promoted the adhesion of Schwann cells but had no such activity on peripheral nervous system neurons and axons. The present study shows that collagen XVIII is, next to perlecan and agrin, the third basal lamina heparan sulfate proteoglycan (HSPG) and the first collagen/proteoglycan with heparan sulfate side chains. By using monoclonal antibodies to an unidentified HSPG in chick, 14 cDNA clones were isolated from a chick yolk sac library. All clones had a common nucleotide sequence that was homologous to the mRNA sequences of mouse and human collagen XVIII. The deduced amino acid sequence of the chick fragment shows an 83% overall homology with the human and mouse collagen XVIII. Similar to the human and mouse homologue, the chick collagen XVIII mRNA has a size of 4.5 kilobase pairs. In Western blots, collagen XVIII appeared as a smear with a molecular mass of 300 kDa. After treatment with heparitinase, the protein was reduced in molecular mass by 120 kDa to a protein core of 180 kDa. Collagen XVIII has typical features of a collagen, such as its existence, under non-denaturing conditions, as a non-covalently linked oligomer, and a sensitivity of the core protein to collagenase digestion. It also has characteristics of an HSPG, such as long heparitinase-sensitive carbohydrate chains and a highly negative net charge. Collagen XVIII is abundant in basal laminae of the retina, epidermis, pia, cardiac and striated muscle, kidney, blood vessels, and lung. In situ hybridization showed that the main expression of collagen XVIII HSPG in the chick embryo is in the kidney and the peripheral nervous system. As a substrate, collagen XVIII moderately promoted the adhesion of Schwann cells but had no such activity on peripheral nervous system neurons and axons. heparan sulfate proteoglycan embryonic day phosphate-buffered saline monoclonal antibody neural cell adhesion molecule. Heparan sulfate proteoglycans (HSPGs)1 are members of a family of cell-surface proteins with long carbohydrate chains of repeating disaccharide units (1Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1012) Google Scholar, 2Wright T.N. Kinsella M.G. Quarnstroem E.E. Curr. Opin. Cell Biol. 1992; 4: 793-801Crossref PubMed Scopus (338) Google Scholar, 3Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (550) Google Scholar). They exist as integral membrane proteins (4Yanagishita M. Hascall V.C. J. Biol. Chem. 1992; 267: 9451-9454Abstract Full Text PDF PubMed Google Scholar) or as secreted extracellular matrix proteins. HSPGs have a variety of biological functions, such as serving as molecular sieves for the ultrafiltration of blood in the kidney (5Kanwar Y. Veis A. Kimura J. Jakubowski M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 762-766Crossref PubMed Scopus (77) Google Scholar), sequestering growth factors (6Vlodavsky I. Folkman J. Sullivan R. Fridman R. Ishai-Michaeli R. Sasse J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2292-2296Crossref PubMed Scopus (841) Google Scholar, 7Saksela O. Moscatelli D. Sommer A. Rifkin D. J. Cell Biol. 1988; 107: 743-751Crossref PubMed Scopus (653) Google Scholar, 8Rapraeger A. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1291) Google Scholar), lining blood vessels (9Kojima T. Leone C. Marchildon G. Marcum J. Rosenberg R. J. Biol. Chem. 1992; 267: 4859-4869Abstract Full Text PDF PubMed Google Scholar), and serving as a structural component of basal laminae. Perlecan, a large proteoglycan from Engelbreth-Holm-Swarm mouse sarcoma, was the first molecularly identified member of the extracellular HSPGs (10Hassell J. Robey P. Barrach H. Wilczek J. Rennard S. Martin G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4494-4498Crossref PubMed Scopus (501) Google Scholar, 11Noonan D.M. Fulle A. Valente P. Cai S. Horigan E. Sasaki M. Yamada Y. Hassell J. J. Biol. Chem. 1991; 266: 22939-22947Abstract Full Text PDF PubMed Google Scholar). cDNA cloning of the murine and human perlecan showed that its protein core consists of various domains that are homologous to the low density lipoprotein receptor, laminin, NCAM, and epidermal growth factor. The perlecan core protein with over 400 kDa is one of the largest gene products known today (11Noonan D.M. Fulle A. Valente P. Cai S. Horigan E. Sasaki M. Yamada Y. Hassell J. J. Biol. Chem. 1991; 266: 22939-22947Abstract Full Text PDF PubMed Google Scholar, 12Murdoch A.D Dodge G.R. Cohen I. Tuan R.S. Iozzo R.V. J. Biol. Chem. 1992; 267: 8544-8557Abstract Full Text PDF PubMed Google Scholar). The second representative member of extracellular HSPGs is agrin (13Tsen G. Halfter W. Kröger S. Cole G.J. J. Biol. Chem. 1995; 270: 3392-3399Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 14Gesemann M. Denzer A.J. Ruegg M.A. J. Cell Biol. 1995; 128: 625-636Crossref PubMed Scopus (207) Google Scholar), a protein that was first isolated from fish electric organ for its activity to cluster acetylcholine receptors on the surface of myoblasts (15Nitkin R.M. Smith M.A. Magill C. Fallon J.R. Yao Y.-M.M.. Wallace B.G. McMahan U.J. J. Cell Biol. 1987; 105: 2471-2478Crossref PubMed Scopus (368) Google Scholar). Like perlecan, agrin is a large multi-domain protein of over 200 kDa with several laminin G, follistatin, and epidermal growth factor-like repeats (16Rupp F. Payan D.G. Magill-Solc C. Cowan D.M. Scheller R.M. Neuron. 1991; 6: 811-823Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 17Tsim K.W.K. Ruegg M.A. Escher G. Kröger S. McMahan U.J. Neuron. 1992; 8: 677-689Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 18Ruegg M.A. Tsim W.K. Horton S.E. Kröger S. Escher G. Gensch E.M. McMahan U.J. Neuron. 1992; 8: 691-699Abstract Full Text PDF PubMed Scopus (224) Google Scholar). Side by side comparison shows that the domain arrangements at the C-terminal part of agrin is similar to that of perlecan (1Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1012) Google Scholar, 19Timpl R. Brown J.C. BioEssays. 1996; 18: 123-132Crossref PubMed Scopus (583) Google Scholar). Members of the collagen family, such as collagen IX and XII, also carry glycosaminoglycan side chains and exist as part-time proteoglycans. So far, however, only chondroitin sulfate chains have been shown to be associated with collagen protein cores (20Fukai N. Apte S. Olsen B.R. Methods Enzymol. 1994; 245: 3-28Crossref PubMed Google Scholar). Here, we present evidence that collagen XVIII is the first member of the collagen family with heparan sulfate side chains. Collagen XVIII was identified several years ago by screening cDNA libraries with probes for collagen-like proteins (21Oh S.P. Yamagata Y. Muragaki Y. Timmons S. Ooshima A. Olsen B.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4229-4233Crossref PubMed Scopus (161) Google Scholar, 22Oh S.P. Warman M.L. Seldin M.F. Cheng S.-D. Knoll J.H.M. Timmons S. Olsen B.R. Genomics. 1994; 19: 494-499Crossref PubMed Scopus (118) Google Scholar, 23Pihlajaniemi T. Rehn M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4234-4238Crossref PubMed Scopus (148) Google Scholar). A characteristic feature of collagen XVIII is its composition of 10 collagenous repeats alternating with 11 non-collagenous repeats (24Saarela J. Ylikarppa R. Rehn M. Purmonen S. Pihlajamniemi T. Matrix Biol. 1998; 16: 319-328Crossref PubMed Scopus (114) Google Scholar). Mouse and human collagen XVIII are expressed in three alternative splice variants and localized at perivascular basal laminae (25Muragaki Y. Timmons S. Griffith C.M. Oh S.P. Fadel B. Quertermous T. Olsen B.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8763-8767Crossref PubMed Scopus (171) Google Scholar, 26Rehn M. Pihlajaniemi T. J. Biol. Chem. 1995; 270: 4705-4711Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 27Rehn M. Hintikka E. Pihlajaniemi T. Genomics. 1996; 32: 436-446Crossref PubMed Scopus (53) Google Scholar). Together with collagen XV, collagen XVIII is a member of a new collagen subfamily, named multiplexins (21Oh S.P. Yamagata Y. Muragaki Y. Timmons S. Ooshima A. Olsen B.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4229-4233Crossref PubMed Scopus (161) Google Scholar). Collagen XVIII came into the focus of medical interest when an 18-kDa anti-angiogenic peptide with tumor-suppressing activity, named endostatin, turned out to be the C-terminal part of collagen XVIII (28O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4250) Google Scholar). Based on the presence of several consensus sequences for glycosaminoglycan attachment, it was suggested that collagen XVIII could be a proteoglycan (21Oh S.P. Yamagata Y. Muragaki Y. Timmons S. Ooshima A. Olsen B.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4229-4233Crossref PubMed Scopus (161) Google Scholar, 24Saarela J. Ylikarppa R. Rehn M. Purmonen S. Pihlajamniemi T. Matrix Biol. 1998; 16: 319-328Crossref PubMed Scopus (114) Google Scholar). However, attempts to show that collagen XVIII is a chondroitin sulfate proteoglycan were unsuccessful, as the digestion with chondroitinase did not lead to a shift in the molecular weight of the protein (25Muragaki Y. Timmons S. Griffith C.M. Oh S.P. Fadel B. Quertermous T. Olsen B.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8763-8767Crossref PubMed Scopus (171) Google Scholar). The present data demonstrate that collagen XVIII is indeed a proteoglycan with almost half of its molecular weight in heparan sulfate side chains. Furthermore, collagen XVIII is a constituent of almost all embryonic and adult basal laminae. The two monoclonal antibodies (mAb 6C4 and 1B11) against a heparitinase-sensitive proteoglycan from chick embryonic basal lamina were obtained from hybridoma clones of X63Ag8.653 myeloma cells (29Kearney J.F. Radbruch A. Liesegang B. Rajewsky K. J. Immunol. 1979; 123: 1548-1558PubMed Google Scholar) fused with splenocytes of a mouse immunized with embryonic day (E) 10 chick retinal basal laminae. Cross-reactivity studies (30Halfter W. Schurer B. Exp. Cell Res. 1994; 214: 285-296Crossref PubMed Scopus (17) Google Scholar) and the isolation of cDNAs with identical nucleotide sequences with both antibodies (this study) show that the 1B11 and 6C4 antibodies recognize the same protein. Monoclonal antibodies to agrin (clone 6D2; see Ref. 31Halfter W. J. Neurosci. 1993; 13: 2863-2873Crossref PubMed Google Scholar), collagen IV (clones IA8, IIB12, ID2, see Ref. 32Fitch J.M. Mentzer A. Mayne R. Linsenmayer T.F. Development. 1989; 105: 85-95PubMed Google Scholar), kindly provided by Drs. Fitch and Linsenmayer (Tufts University, Boston), NCAM (clone 9H2; see Ref. 33Halfter W. Schurer B. Yip J.W. Yip L.Y.P. Tsen G. Lee J.A. Cole G.J. J. Comp. Neurol. 1997; 383: 1-17Crossref PubMed Scopus (76) Google Scholar), and tenascin (clone M1, see Ref.34, Developmental Studies Hybridoma Bank, Johns Hopkins University, Baltimore) were mouse IgGs. Rabbit antisera to Engelbreth-Holm-Swarm mouse tumor perlecan (10Hassell J. Robey P. Barrach H. Wilczek J. Rennard S. Martin G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4494-4498Crossref PubMed Scopus (501) Google Scholar, 35Vigny M. Ollier-Hartmann M. Lavigne M. Fayein N. Jeanny J. Laurent M. Courtois Y. J. Cell. Physiol. 1988; 137: 321-328Crossref PubMed Scopus (142) Google Scholar) were a generous gift from Dr. M. Vigny (Institute National de la Sante, Paris) and Dr. J. Hassel (Shriners Hospital for Crippled Children, Tampa Bay, FL). A mouse monoclonal antibody (clone 33-2, see Ref. 36Bayne E. Anderson M. Fambrough D. J. Cell Biol. 1984; 99: 1486-1501Crossref PubMed Scopus (145) Google Scholar) to a chicken heparan sulfate proteoglycan was obtained from the Developmental Studies Hybridoma Bank (John Hopkins University, Baltimore). The tissue distribution and the molecular weight of the antigen strongly suggest that the 33-2 antibody recognizes chick perlecan. The 6C4 and 1B11 hybridoma supernatant was used to screen each 600,000 and 300,000 recombinants from an E5 chick yolk sac lambda-ZAP II cDNA expression library (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Eleven positive phage clones from the 6C4 screen and 3 clones from the 1B11 screen were plaque-purified, and Bluescript II SK(+) phagemids containing the inserts were rescued by in vivo excision. The plasmids were purified using the Wizard plasmid purification kit (Promega, Madison, WI) and sequenced on both strands at the University of Pittsburgh sequencing facility by using T3, T7, and internal sequencing primers and the Dye Terminator Cycle Sequencing Ready Reaction Kit (ABI, Foster City, CA). Sequences were aligned and a contig was constructed using the “Sequencer” software (Gene Codes Inc., Ann Arbor, MI). The sequences were compared with published data base banks using the Blast search algorithm (37Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59917) Google Scholar). DNA sequences were translated into amino acid sequences using the Sequencer program, and the amino acid sequence was aligned with the human and mouse collagen XVIII peptide sequences using the P-Blast program (37Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59917) Google Scholar). For Northern blots, mRNA from E10 chick brain, liver, and kidney was isolated using the “Total RNA Isolation” kit (Promega) followed by using the “Poly(A) Track” kit (Promega). One μg of poly(A)+ mRNA samples were separated in 1% agarose gels containing 2.2 mformaldehyde, transferred to positively charged nylon membranes (Immobilon N, Millipore), and cross-linked with UV (Stratagene). cRNA hybridization probes were synthesized from the linearized p10d template in the presence of digoxigenin UTP using the RNA-polymerase labeling kit (Boehringer Mannheim) according to the manufacturer's instructions. The blots were developed using a chemiluminescence kit (Boehringer Mannheim). A digoxigenin-labeled RNA ladder (Boehringer Mannheim) was used to determine the size of the collagen XVIII mRNA. Another set of Northern blots, containing mRNA samples of E9 chick brain, gut, heart, liver, and cultured astrocytes (38Tsen G. Napier A. Halfter W. Cole G.J. J. Biol. Chem. 1995; 270: 15934-15937Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), was probed with the radiolabeled insert from the p10d plasmid. The probe was synthesized using random primers and [α-32P]dATP. Membranes were hybridized for 20 h with radioactive probe and washed with high stringency. Amnion, kidneys, and meninges were dissected from E10 chick embryos and homogenized in calcium- and magnesium-free Hanks' solution (CMF), 50 μg/ml DNase I (Sigma). The homogenates were spun at 3000 rpm for 5 min, and the pellets were re-extracted in 8 m urea in CMF and centrifuged again at 3000 rpm. The supernatants from the urea extracts were used as the samples. To characterize further the biochemical nature of the protein, samples were treated with 0.5 units of heparitinase (Seikagaku, Rockville, MA), 0.5 units of chondroitinase ABC (Sigma), or 5 units/ml collagenase (Sigma type VII) for 1 h at 37 °C in PBS, 2% bovine serum albumin, pH 7.4. Intact tissues were treated first with the enzymes for 1 h and washed in CMF twice. Following homogenization in CMF and centrifugation, the pellets were extracted in 8 m urea, spun again, and the supernatants from the urea extracts were used as samples. Vitreous bodies were collected from E10 chick eyes, centrifuged at 15,000 rpm, and the supernatant was used, undigested or digested, as samples. Vitreous body supernatant and tissue extracts were mixed with SDS sample buffer/β-mercaptoethanol and were loaded either boiled or non-boiled onto the gels. The proteins were separated by 3.6–14% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon P membrane filters (Millipore, Bedford, MA). After blocking with 5% skim milk in CMF, the blots were incubated with the 6C4 or 1B11 hybridoma supernatants for 1 h. The blots were rinsed three times in CMF/milk, incubated with 1:5000 diluted (CMF/milk) alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) for 1 h, and finally developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as described previously (31Halfter W. J. Neurosci. 1993; 13: 2863-2873Crossref PubMed Google Scholar). To detect any nonspecific proteolysis during the enzyme treatments, blots with heparitinase- and collagenase-treated samples were also stained with a monoclonal antibody to tenascin (clone M1, see above). The trunks of E3 to E16 chick embryos and the dissected spinal cord of a 5-day-old chick (P5) were fixed in 4% paraformaldehyde in 0.1 m potassium phosphate buffer, pH 7.4, for 1 h. After washing in CMF and cryoprotecting with 30% sucrose for 4 h, the specimens were embedded in Tissue-Tek (Miles, Elkhart, IN) and sectioned with a cryostat at 25 μm. Muscle samples from post-hatched (P) 5 chicken were sectioned unfixed. Sections were mounted on Superfrost slides (Fisher) and incubated with hybridoma supernatants for 1 h. After 3 rinses, the sections were incubated with 1:500 Cy3-labeled goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) for another hour. After 2 final rinses, the specimens were examined with an epifluorescence microscope (Zeiss, Thornwood, NY). Basal lamina whole mounts from E6 chick retinae were prepared as described (39Halfter W. Reckhaus W. Kroeger S. J. Neurosci. 1987; 7: 3712-3722Crossref PubMed Google Scholar). The whole mounts were rinsed several times with 2% Triton X-100 to remove all cellular components and stained with the 6C4 mAb to collagen XVIII or the 9H2 mAb to NCAM (see above). The p10d plasmid was linearized, and a digoxigenin-labeled antisense and sense cRNA probe were synthesized from the template using an RNA polymerase labeling kit (Boehringer Mannheim). The in situ hybridization procedure of tissue sections follows the procedure described previously (40Schaeren-Wiemers N. Gerfin-Moser A. Histochemistry. 1993; 100: 431-440Crossref PubMed Scopus (1085) Google Scholar). Adjacent sections were also labeled with probes to agrin and collagen XII. The isolation of the agrin plasmid has been described previously (13Tsen G. Halfter W. Kröger S. Cole G.J. J. Biol. Chem. 1995; 270: 3392-3399Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), and the plasmid to collagen XII was obtained by antibody screening of a E5 chick yolk sac cDNA library with a polyclonal antiserum to chondroitin sulfate proteoglycans from chick embryos. The identity of the collagen XII clone was confirmed by matching the DNA sequence with the mRNA sequence of collagen XII (bp 5070–5604) in the data bank using the Blast search. 2W. Halfter, unpublished observations. Collagen XVIII was isolated from E10 chicken vitreous bodies by ion exchange chromatography followed by immunoaffinity purification using the 6C4 mAb coupled to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech, see Ref.30Halfter W. Schurer B. Exp. Cell Res. 1994; 214: 285-296Crossref PubMed Scopus (17) Google Scholar). The vitreous bodies from approximately 400 eyes were collected and spun at 15,000 rpm for 20 min. The supernatant (100 ml) was run over a 3-ml column of Q-Sepharose (Amersham Pharmacia Biotech). The column was washed with 50 mm Tris, pH 7.4, and eluted with a salt gradient from 0 to 2 m NaCl in 50 mm Tris, pH 7.4. Fractions containing collagen XVIII immunoreactivity were collected, dialyzed against CMF, and run over an affinity column containing 6C4 mAb conjugated to Sepharose. After extensive washing with CMF, 0.5% Tween, and CMF alone, collagen XVIII was eluted with 0.1 m diethylamine buffer, pH 11.5. Fractions were tested by a dot assay on nitrocellulose membrane filters, and collagen XVIII-containing fractions were concentrated using Centricon filters (Millipore). The concentration of the proteoglycan was estimated both by the dye-binding method of Minamide and Bamburg (41Minamide L.S. Bamburg J.R. Anal. Biochem. 1990; 190: 66-70Crossref PubMed Scopus (237) Google Scholar) and by gel electrophoresis. Gels were stained with Coomassie and a modified Alcian blue/silver staining procedure for the detection of proteoglycans (42Moller H.J. Heinegard D. Poulsen J.H. Anal. Biochem. 1993; 209: 169-175Crossref PubMed Scopus (87) Google Scholar). The concentration of the samples was also estimated by comparing the staining density of the proteoglycan band with that of known concentrations of laminin and fibronectin (Life Technologies, Inc.) in adjacent lanes of the gel. Each purification yielded between 10 and 50 μg of proteoglycan. As substrates for cell adhesion and neurite outgrowth assays, collagen XVIII (50 μg/ml), merosin (Life Technologies, Inc., 50 μg/ml), and ovalbumin (Sigma, 50 μg/ml) were spotted onto nitrocellulose-coated dishes (43Lagenaur C. Lemmon V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7753-7757Crossref PubMed Scopus (395) Google Scholar). The dishes were rinsed 3 times in CMF, 2% ovalbumin, and 60 μl of a cell suspension from trypsin-dissociated E12 chick dorsal root ganglia in Dulbecco's modified Eagle's medium; 2% ovalbumin was added to the substrates. After 6 h, the cultures were rinsed 3 times with Dulbecco's modified Eagle's medium, 2% ovalbumin, and finally fixed in 4% paraformaldehyde for 1 h. The number of cells per microscopic field were counted at 3 different areas of every culture dish. For each substrate, the assays was run in triplicate. To account for variations between different test runs, the average number of cells on the ovalbumin control substrate was set at 100%. The average number of cells attached on the other substrates was expressed as multiples of the ovalbumin control. The identity of the cells as Schwann cells was done by staining cultures with a mAb to P0 (clone 1E8, kindly provided by Dr. E. Frank, University of Pittsburgh, see Ref. 44Bhattacharyya A. Frank E. Ratner N. Brackebury R. Neuron. 1991; 7: 831-844Abstract Full Text PDF PubMed Scopus (95) Google Scholar), a protein specific for Schwann cells. According to the immunohistochemistry, over 90% of the cells in the ovalbumin and collagen XVIII-coated dishes were Schwann cells. Two monoclonal antibodies to a putatively new basal lamina HSPG were used to screen an E5 chick yolk sac cDNA expression library. The screen resulted in the isolation of 14 cDNA clones ranging in size from 2 to 2.5 kb. All 14 clones overlapped in their sequence. A Blast search revealed that the common sequence was highly homologous to the 3′ third of human and mouse collagen XVIII mRNA. The 2.5-kb p10d cDNA clone was the longest and most complete cDNA clone, as it contained at its 3′ end an AATAAA termination signal followed by a poly(A) tail (Gene Bank™ accession number AF083440). The p10d clone also contained 1.2 kb of 3′-untranslated region and 1.2 kb of a coding sequence that was homologous to the 3′ third of the coding region of human and mouse collagen XVIII. Translation of the coding sequence showed a 387-amino acid long peptide that started at its N-terminal end with a typical collagen sequence that was followed by a short, non-collagenous stretch. After yet another short collagen sequence, the peptide ended at its C-terminal part with a long, non-collagenous domain (Fig. 1). The two chick collagen domains were over 90% homologous with the 9th and 10th collagen domain (Col9 and Col10) of the human and mouse collagen XVIII. Both non-collagenous domains (NC10 and NC11) were between 60 and 90% identical to the human and mouse collagen XVIII counterparts. The most homologous sequence in the NC11 domain corresponded to the N-terminal part of the 18-kDa endostatin peptide (Fig. 1). The overall amino acid sequence identity of the chick and human collagen XVIII was 65%, and the overall homology was 83%. The identity and homology between the chick and human amino acid sequences are slightly lower than the identity and homology between the human and mouse collagen XVIII amino acid sequences, which are 79 and 95%, respectively. The chick sequence showed one serine-glycine consensus sequence as a potential GAG attachment site, which was conserved in the human and mouse homologue. Northern blot showed that the chick collagen XVIII was abundant in kidney and heart and barely detectable in liver and brain tissue. The message size was in all tissues 4.5 kb (Fig. 2, lanes 1–4). The Northern blots were repeated but using a radiolabeled DNA probe. The blots showed a single band of 4.5 kb in all samples that was faint in samples of brain (Fig. 2, lane 6), gut, and liver (not shown) but prominent in samples from cultured astrocytes (Fig. 2, lane 5). Based on the overall sequence homology, we assumed that the chick HSPG is the chick homologue of collagen XVIII, and we decided to tentatively refer to the unidentified chick HSPG as collagen XVIII in further studies.Figure 2Northern blots showing the size and abundance of chick collagen XVIII mRNA in kidney (lane 1), liver (lane 2), brain (lane 3), and heart (lane 4). A strong band of 4.5 kb was detected in samples of kidney and heart, whereas faint bands were detected in liver and brain. The blot with lanes 1–4 was probed with a digoxigenin-labeled RNA. Another blot, probed with a radiolabeled DNA probe (lanes 5 and 6), showed a strong band of 4.5 kb in a sample from cultured astrocytes and a faint 4.5-kb band in an mRNA sample of brain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Western blots, intact chick collagen XVIII from chick vitreous body appeared as a smear with an apparent molecular mass of 300 kDa (Fig. 3, lane 1, and Fig. 4, lane 4). Digestion with heparitinase led to a reduction of the size of the protein by 120 kDa to a core protein of 180 kDa (Fig. 3, lane 2, and Fig. 4,lane 5). Treatment with chondroitinase had no effect on the size or banding pattern of the molecule (Fig. 3, lane 4, and Fig. 4, lane 6), confirming previous experiments with mouse collagen XVIII (25Muragaki Y. Timmons S. Griffith C.M. Oh S.P. Fadel B. Quertermous T. Olsen B.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8763-8767Crossref PubMed Scopus (171) Google Scholar). When the samples were treated with collagenase, the protein was no longer detectable in Western blots (Fig. 3,lane 3, and Fig. 4, lane 7), demonstrating a collagenase-sensitive core protein. To check the collagenase and heparitinase preparations for potential protease contamination, the same samples that were used to detect collagen XVIII were also probed with an antibody to tenascin. Tenascin, a large 220-kDa glycoprotein, was degraded neither by collagenase (Fig. 3, lane 6) nor heparitinase (Fig. 3, lane 7). The specificity of collagenase for only collagenous proteins was confirmed in an independent study by digesting chick vitreous body and meninges with the enzyme (45Halfter W. J. Comp. Neurol. 1998; 397: 89-104Crossref PubMed Scopus (46) Google Scholar, 46Halfter W. Schurer B. J. Comp. Neurol. 1998; 397: 105-117Crossref PubMed Scopus (17) Google Scholar); all collagenous components from vitreous body and meninges, such as collagen I, II, IV, and IX, were no longer detectable in Western blots, whereas the abundance and the banding pattern of non-collagenous vitreous body and meningeal proteins, such as laminin, agrin, nidogen, tenascin, fibronectin, and axonin I, were unchanged.Figure 4Western blots comparing the banding pattern of collagen XVIII in non-boiled (lanes 1–3) and boiled samples (lanes 4–7) from chick vitreous body. The samples in lanes 2 and 5 were treated with heparitinase, the samples in lane 3 and 6 with chondroitinase, and lane 7 with collagenase. The binding of collagen XVIII to anion exchange beads is shown in lanes 8–12. A sample of vitreous body (lane 8) was incubated with Q-Sepharose. After 1 h, collagen XVIII was no longer detectab" @default.
• W2005294501 created "2016-06-24" @default.
• W2005294501 creator A5014007420 @default.
• W2005294501 creator A5063503729 @default.
• W2005294501 creator A5070406068 @default.
• W2005294501 creator A5072344748 @default.
• W2005294501 date "1998-09-01" @default.
• W2005294501 modified "2023-10-05" @default.
• W2005294501 title "Collagen XVIII Is a Basement Membrane Heparan Sulfate Proteoglycan" @default.
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