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• W2018096448 abstract "Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The α1(XV) chain contains a highly interrupted collagenous region of 577 residues, and noncollagenous amino- and carboxyl-terminal domains of 530 and 256 residues, respectively. Cysteines are present in each domain and consensus sequences forO-linked glycosaminoglycans are situated in the amino terminus and in two large, noncollagenous interruptions. We now report that type XV collagen is a chondroitin sulfate proteoglycan in human tissues and cultured cells, and that the α chains are covalently linked by interchain disulfide bonds only between the two cysteines in the collagenous region. Western blotting of tissue extracts revealed a diffuse smear with a mean size ≥400 kDa, which after chondroitinase digestion resolved into a 250-kDa band in umbilical cord, and 250- and 225-kDa bands in placenta, lung, colon, and skeletal muscle. The latter two bands were also directly visualized by alcian blue/silver staining of a purified placenta extract. In a human rhabdomyosarcoma cell line, almost all of the newly synthesized type XV collagen was secreted into the medium and upon chondroitinase digestion just the 250-kDa α chain was generated. Chondroitinase plus collagenase digestion of tissue and medium proteins followed by Western blotting using domain-specific antibodies revealed a 135-kDa amino-terminal fragment containing glycosaminoglycan chains and a 27-kDa fragment representing the intact carboxyl terminus. However, a truncated carboxyl peptide of ∼8-kDa was also evident in tissue extracts containing the 225-kDa form. Our data suggest that the 225-kDa form arises from differential carboxyl cleavage of the 250-kDa form, and could explain the ∼19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and Ständker, L. (1999)Biochemistry 38, 10217–10224), which may be liberated from the α1(XV) chain. Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The α1(XV) chain contains a highly interrupted collagenous region of 577 residues, and noncollagenous amino- and carboxyl-terminal domains of 530 and 256 residues, respectively. Cysteines are present in each domain and consensus sequences forO-linked glycosaminoglycans are situated in the amino terminus and in two large, noncollagenous interruptions. We now report that type XV collagen is a chondroitin sulfate proteoglycan in human tissues and cultured cells, and that the α chains are covalently linked by interchain disulfide bonds only between the two cysteines in the collagenous region. Western blotting of tissue extracts revealed a diffuse smear with a mean size ≥400 kDa, which after chondroitinase digestion resolved into a 250-kDa band in umbilical cord, and 250- and 225-kDa bands in placenta, lung, colon, and skeletal muscle. The latter two bands were also directly visualized by alcian blue/silver staining of a purified placenta extract. In a human rhabdomyosarcoma cell line, almost all of the newly synthesized type XV collagen was secreted into the medium and upon chondroitinase digestion just the 250-kDa α chain was generated. Chondroitinase plus collagenase digestion of tissue and medium proteins followed by Western blotting using domain-specific antibodies revealed a 135-kDa amino-terminal fragment containing glycosaminoglycan chains and a 27-kDa fragment representing the intact carboxyl terminus. However, a truncated carboxyl peptide of ∼8-kDa was also evident in tissue extracts containing the 225-kDa form. Our data suggest that the 225-kDa form arises from differential carboxyl cleavage of the 250-kDa form, and could explain the ∼19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and Ständker, L. (1999)Biochemistry 38, 10217–10224), which may be liberated from the α1(XV) chain. basement membrane zone antibody recognizing the type XV carboxyl-terminal domain antibody recognizing the type XV amino-terminal domain fibril-associated collagens with interrupted triple helices, PMSF, phenylmethylsulfonyl fluoride N-ethylmaleimide 3-[(cholamidopropyl)dimethylammoniol]-1-propanesulfonate dithiothreitol polyacrylamide gel electrophoresis glycosaminoglycan N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Basement membrane zones (BMZs)1 can be operationally defined as morphological entities consisting of a basement membrane plus its closely associated matrix components, which extend into or originate from the sub-basal lamina (1.Adachi E. Hopkinson I. Hayashi T. Int. Rev. Cytol. 1997; 173: 73-156Crossref PubMed Google Scholar). The BMZ contains the molecules responsible for attaching basement membranes to their contiguous stroma and/or epithelium. Those components integral to basement membranes include type IV collagen, laminin, entactin/nidogen, and perlecan (for review, see Ref. 2.Paulsson M. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 93-127Crossref PubMed Scopus (470) Google Scholar), whereas a plethora of other matrix proteins, glycoproteins, and proteoglycans have been assigned to the BMZ using different methods. Many of these constituents have been studied in depth biochemically and ultrastructurally, while several newer and less abundant ones are not yet well characterized. Within this latter category are three more recently discovered nonfibrillar collagens: types XV, XVIII, and XIX (3.Myers J.C. Kivirikko S. Gordon M.K. Dion A.S. Pihlajaniemi T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10144-10148Crossref PubMed Scopus (65) Google Scholar, 4.Oh S.P. Kamagata 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, 5.Rehn M. Pihlajaniemi T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4234-4238Crossref PubMed Scopus (148) Google Scholar, 6.Myers J.C. Yang H.G. D'Ippolito J.A. Presente A. Miller M.K. Dion A.S. J. Biol. Chem. 1994; 269: 18549-18557Abstract Full Text PDF PubMed Google Scholar). Independently identified from DNA clone isolation, they are considered members of a unique collagen subclass because of their widespread distribution in BMZs of many tissues (7.Myers J.C. Li D. Bageris A. Abraham V. Dion A.S. Amenta P.S. Am. J. Pathol. 1997; 151: 1729-1740PubMed Google Scholar). Immunohistochemical light microscopy demonstrated that these three collagens co-localize in some BMZs, but are differentially expressed in others (7.Myers J.C. Li D. Bageris A. Abraham V. Dion A.S. Amenta P.S. Am. J. Pathol. 1997; 151: 1729-1740PubMed Google Scholar, 8.Muragaki 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, 9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar, 10.Hägg P.M. Hägg P.O. Peltonen S. Autio-Harmainen H. Pihlajaniemi T. Am. J. Pathol. 1997; 150: 2075-2086PubMed Google Scholar, 11.Saarela J. Rehn M. Oikarinen A. Autio-Harmainen H. Pihlajaniemi T. Am. J. Pathol. 1998; 153: 611-626Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 12.Halfter W. Dong S. Schurer B. Cole G.J. J. Biol. Chem. 1998; 273: 25404-25412Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Type XV and XVIII, but not type XIX, collagens were also shown to exhibit major similarities by primary structure alignment. Comparison of domain arrangement, restricted sequence homology, as well as intron/exon organization indicated that α1(XV) and α1(XVIII) evolved from a common ancestral gene (4.Oh S.P. Kamagata 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, 13.Kivirikko S. Heinämäki P. Rehn M. Honkanen N. Myers J.C. Pihlajaniemi T. J. Biol. Chem. 1994; 269: 4773-4779Abstract Full Text PDF PubMed Google Scholar, 14.Muragaki Y. Abe N. Ninomiya Y. Olsen B.R. Ooshima A. J. Biol. Chem. 1994; 269: 4042-4046Abstract Full Text PDF PubMed Google Scholar, 15.Rehn M. Hintikka E. Pihlajaniemi T. J. Biol. Chem. 1994; 269: 13929-13935Abstract Full Text PDF PubMed Google Scholar, 16.Hägg P.M. Muona A. Liétard J. Kivirikko S. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 17824-177831Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Both collagens, but especially type XV, contain extensive interruptions in their collagenous regions such that the majority of the residues in each chain are found within the amino- and carboxyl-terminal noncollagenous domains. In particular, the carboxyl-terminal domain of type XVIII collagen has become a focal point in tumor biology upon finding that the terminal 20-kDa fragment is identical to the potent anti-angiogenic factor, endostatin (17.O'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 (4263) Google Scholar). Pursuant to this discovery is more current research in which the analogous peptide of type XV collagen, displaying by far the highest degree of sequence conservation with type XVIII, is being investigated for related properties (18.Ramchandran R. Dhanabal M. Volk R. Waterman M.J.F. Segal M. Lu H. Knebelmann B. Sukhatme V.P. Biochem. Biophys. Res. Commun. 1999; 255: 735-739Crossref PubMed Scopus (245) Google Scholar, 19.John H. Preissner K.T. Forssmann W.-G. Ständker L. Biochemistry U. S. A. 1999; 38: 10217-10224Crossref Scopus (59) Google Scholar). Another parallel between types XV and XVIII collagen can be drawn by comparing their noncollagenous amino-terminal domains. Among the common features are a number of consensus sequences for attachment ofO-linked glycosaminoglycans (4.Oh S.P. Kamagata 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, 5.Rehn M. Pihlajaniemi T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4234-4238Crossref PubMed Scopus (148) Google Scholar, 13.Kivirikko S. Heinämäki P. Rehn M. Honkanen N. Myers J.C. Pihlajaniemi T. J. Biol. Chem. 1994; 269: 4773-4779Abstract Full Text PDF PubMed Google Scholar, 14.Muragaki Y. Abe N. Ninomiya Y. Olsen B.R. Ooshima A. J. Biol. Chem. 1994; 269: 4042-4046Abstract Full Text PDF PubMed Google Scholar). (Several additional sequences are present within interruptions in the collagenous region (see Refs. 3.Myers J.C. Kivirikko S. Gordon M.K. Dion A.S. Pihlajaniemi T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10144-10148Crossref PubMed Scopus (65) Google Scholar, 4.Oh S.P. Kamagata 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, and 6.Myers J.C. Yang H.G. D'Ippolito J.A. Presente A. Miller M.K. Dion A.S. J. Biol. Chem. 1994; 269: 18549-18557Abstract Full Text PDF PubMed Google Scholar).). Until recently, it was not known whether any of these sites were occupied. It has since been described that α1(XVIII) chains are sensitive to heparitinase, but not to chondroitinase ABC digestion (12.Halfter W. Dong S. Schurer B. Cole G.J. J. Biol. Chem. 1998; 273: 25404-25412Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Thus, type XVIII collagen is a heparan sulfate proteoglycan with a core protein with molecular mass of 180 kDa. The first immunochemical studies of type XV collagen to determine its tissue distribution were reported using antibodies directed at the carboxyl terminus of the protein (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar). In human placenta and colon tissue extracts, our antibody, derived from a recombinant protein antigen, recognized a 116-kDa collagenase-sensitive protein and a 27-kDa collagenase-resistant fragment (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar). The latter was in accord with the size expected for the 256-residue carboxyl terminus, whereas the former appeared considerably smaller than would be predicted for the 1388-residue intact protein. In a separate analysis, other investigators showed that their type XV antibody, generated from a synthetic carboxyl peptide, reacted with a 110-kDa band in human heart extract and with 110- and 70-kDa bands in kidney samples (10.Hägg P.M. Hägg P.O. Peltonen S. Autio-Harmainen H. Pihlajaniemi T. Am. J. Pathol. 1997; 150: 2075-2086PubMed Google Scholar). Subsequent use of antibodies from both sources, however, revealed a similar pattern of BMZ localization (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar, 10.Hägg P.M. Hägg P.O. Peltonen S. Autio-Harmainen H. Pihlajaniemi T. Am. J. Pathol. 1997; 150: 2075-2086PubMed Google Scholar). To conduct a comprehensive biochemical characterization of type XV collagen, we embarked upon a series of purification steps beginning with human placenta tissue. Type XV was identified using both carboxyl-terminal antibodies and a new antibody prepared against a peptide sequence located in the amino-terminal noncollagenous domain. The results presented here surprisingly show that type XV collagen exists as a chondroitin sulfate proteoglycan in the five human tissues examined. In four of these tissues, type XV is present as 250- and 225-kDa core protein forms, which differ at their carboxyl terminus.In vitro studies of type XV production in cultured human cells showed that almost all of the newly synthesized collagen is secreted into the medium, consists of only the 250-kDa core protein form, and is modified by the addition of chondroitin sulfate chains. Further analysis of type XV collagen by differential use of the domain-specific antibodies revealed that the trimer is linked by interchain disulfide bonds and these involve only two of the eight cysteines in the molecule. Taken together, our data provide crucial new insight into the structure and expression of this complex BMZ collagen. Preparation of the type XV collagen recombinant protein (corresponding to the first 120 residues of the noncollagenous carboxyl-terminal domain) and the original type XV-COOH antibody (COOH-Ab) have been described previously (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar). Sera from the three additional rabbits injected (Berkeley Antibody Co., Richmond, CA) with the carboxyl recombinant protein (500 μg/rabbit) were precipitated by addition of 50% saturated ammonium sulfate, dialyzed against phosphate-buffered saline and affinity-purified using Affi-Gel 10 resin according to the manufacturer's instructions (Bio-Rad). The type XV NH2-antibody (NH2-Ab) was prepared using a 15-amino acid peptide (GPGDEEDLAAATTEE) synthesized by the Protein Chemistry Laboratory of the University of Pennsylvania School of Medicine. The peptide was coupled to keyhole limpet hemocyanin and injected into two rabbits. Serum was affinity-purified as above using Affi-Gel 15 resin. The CCL136 human rhabdomyosarcoma cell line was obtained from the American Type Culture Collection and grown in a humidified atmosphere of 5% CO2 at 37 °C. Cells in T75 flasks were grown for 2 days in RPMI medium 1640 containing 10% fetal bovine serum (Sigma) to 90% confluence and used to seed five T75 flasks at a density of ∼7.5 × 106 cells/flask (∼100,000 cells/cm2) in growth medium pre-equilibrated in the CO2 incubator. After 22 h, the cells at ∼75% confluence were washed twice with medium to remove the serum, and were then incubated in medium containing 0.1% serum and 50 μg/ml ascorbate. The following day, the cultures had reached ∼90% confluence. Ascorbate was again added to a final concentration of 50 μg/ml (to the preexisting medium), and cultures were maintained for an additional 20 h. The respective media and cell layer/matrix fractions from the five flasks were pooled and processed as described in the following section. Medium from the cultures (10 ml/flask) was quick-chilled in an ice slurry and adjusted to a final concentration of 5 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride (PMSF), and 10 mm N-ethylmaleimide (NEM). The medium was clarified for 10 min at 7500 × g and concentrated 25–35 fold at 2800 × g at 4 °C using Centriplus XM-100 filters (Amicon/Millipore). Preparation of cell/matrix samples has been described previously (20.Myers J.C. Li D. Rubinstein N.A. Clark C.C. Exp. Cell Res. 1999; 253: 587-598Crossref PubMed Scopus (22) Google Scholar). Cell/matrix and medium samples were aliquoted in small volumes, quick-frozen, and stored at −75 °C. Normal human tissue samples, obtained from the Hospital of the University of Pennsylvania and the Cooperative Human Tissue Network, were frozen at −75 °C or in liquid nitrogen, normally within 60 min after excision. Tissue (∼0.25–0.5 g) was homogenized for 5 × 1 min at 30,000 rpm in an ice-chilled buffer consisting of 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.2 mm PMSF, 22 mm NEM, 0.0162 trypsin inhibitor units/ml aprotinin, and 20 μg/ml leupeptin. The mixture was placed on a rocker platform at 4 °C for 30 min and centrifuged for 10 min at 1500 ×g. Small aliquots of the supernatant were stored at −75 °C. Fresh human placenta (60 g wet weight) was washed several times in sterile ice-chilled 50 mm Tris-HCl, 4.5 m NaCl, 20 mm EDTA, pH 7.5, 1 mm PMSF, 2 mmNEM, 1 μg/ml pepstatin A and stored at −75 °C. Frozen tissue was ground to a powder in a mortar and pestle chilled in liquid nitrogen. The tissue powder was added to a urea-containing extraction buffer in a 5–10:1 (v/w) ratio of buffer to tissue under non-reducing conditions. The buffer consisted of 7 m urea, 50 mmTris-HCl, pH 8.5, 1 mm EDTA, 50 mm NaCl, 2% CHAPS (Sigma) (21.Byers S. Hopkins T.J. Kuettner K.E. Kimura J.H. J. Biol. Chem. 1987; 262: 9166-9174Abstract Full Text PDF PubMed Google Scholar) and the protease inhibitors 100 mmε-amino-n-caproic acid, 10 mm NEM, and 1 mm PMSF. (Extraction in 4 m guanidine HCl, pH 5.8, buffer was equally effective.) The tissue suspension was stirred at 4 °C for 24 h and centrifuged at 32,000 × gfor 20 min. The solubilized protein (100 mg) was incubated with 15 ml of Q-Sepharose Fast Flow resin (Amersham Pharmacia Biotech) pre-equilibrated in 7 m urea, 50 mm Tris-HCl, pH 8.5, 1 mm EDTA, 50 mm NaCl, plus the above protease inhibitors. The sample was incubated with the resin on a rocker overnight at 4 °C. The suspension was centrifuged at 480 × g and the resin washed first with binding buffer containing 0.2% CHAPS; second, with the same buffer containing 0.5m NaCl; and third, with the same buffer containing 1m NaCl. To measure what remained bound, an aliquot of the resin was subsequently boiled in SDS-polyacrylamide gel buffer. All fractions were analyzed by Western blotting. Type XV collagen eluted in 1 m NaCl buffer and was concentrated by ultrafiltration using Centriplus XM-100 filters. The concentrated sample was brought to a final concentration of 100 mm DTT and applied to a 1 × 115-cm column of Sephacryl S-500 Superfine (Amersham Pharmacia Biotech) equilibrated in 50 mm Tris-HCl, pH 8.0, 0.25m sodium sulfate, 20 mm EDTA, and 4m guanidine HCl (22.Clark C.C. Richards C.F. Pacifici M. Iozzo R.V. J. Biol. Chem. 1987; 262: 10229-10238Abstract Full Text PDF PubMed Google Scholar). Fractions were eluted and assayed by dot-blot screening, and the peak fractions were pooled and concentrated by ultrafiltration. Bacterial collagenase (Advance Biofactures, Lynbrook, NY) digestions have been described previously (7.Myers J.C. Li D. Bageris A. Abraham V. Dion A.S. Amenta P.S. Am. J. Pathol. 1997; 151: 1729-1740PubMed Google Scholar, 9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar). Chondroitinase ABC, protease-free (Proteus vulgaris) and heparitinase (Flavobacterium heparinum) were purchased from Seikagaku Corp. (Rockville, MD). Chondroitinase digestions (15 μl) were performed for 90 min at 37 °C in 100 mm Tris-HCl, 30 mm sodium acetate buffer, pH 8.0, using 20 milliunits of enzyme. In those reactions where collagenase digestion was sequentially performed, 4 μl of 50 mm calcium acetate was added, followed by 3 μl (3 units) of bacterial collagenase. Reactions were then incubated for another 60 min at 37 °C. Heparitinase digestions (15 μl) were carried out for 90 min at 37 °C in a pH 7.0 buffer consisting of 100 mm sodium acetate, 10 mm calcium acetate, using 5 milliunits of enzyme. In those reactions where bacterial collagenase digestion was sequentially performed, 4 μl of 5× collagenase buffer (250 mm Tris-HCl, pH 7.2, 50 mm calcium acetate) was added, followed by 3 μl (3 units) of bacterial collagenase. Reactions were then incubated for another 60 min at 37 °C. In all procedures, the control digestion(s), indicated in the specific figure legend, was incubated in the identical buffer and for the identical time and temperature except without the respective enzyme(s). Proteins were transferred from 5–12% polyacrylamide-SDS gels (acrylamide concentrations are specified in the figures and figure legends) to Immobilon-P membranes (Millipore Corp., Marlborough, MA) as described previously (7.Myers J.C. Li D. Bageris A. Abraham V. Dion A.S. Amenta P.S. Am. J. Pathol. 1997; 151: 1729-1740PubMed Google Scholar, 9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar). In the one instance where the 18% polyacrylamide-Tricine gel was used, proteins were transferred to Immobilon-PSQ (Millipore) in 25 mm Tris, 192 mm glycine, and 37.5% methanol for 90 min at 65 V. Secondary antibodies, horseradish peroxidase -linked donkey anti-rabbit Ig (whole antibody or F(ab′)2fragment), obtained from Amersham Pharmacia Biotech, were used at 1:18,000 or 1:2500 dilutions, respectively. Primary antibodies were used at dilutions of 1:500 to 1:2000. The procedure was modified from the 20 steps listed in the published report (23.Møller H.J. Heinegård D. Poulsen J.H. Anal. Biochem. 1993; 209: 169-175Crossref PubMed Scopus (87) Google Scholar). The major differences involved changes in the following steps (S): S1, overnight; S4, 45 min; S5–S7 10 min each; S8 and S9, 1 h each (or until the background cleared); S10, 10 min; S15, 20 min. Additionally, S15 to S20 were conducted at room temperature. Following preparation of our initial type XV antibody (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar), three additional carboxyl-derived antibodies (COOH-Abs) were generated using the same recombinant protein as antigen. Immunostaining of human tissues using the new type XV antibodies (7.Myers J.C. Li D. Bageris A. Abraham V. Dion A.S. Amenta P.S. Am. J. Pathol. 1997; 151: 1729-1740PubMed Google Scholar, 24.Amenta P.S. Briggs K. Xu K. Gamboa E. Jukkola A.F. Li D. Myers J.C. Hum. Pathol. 2000; 31: 359-366Crossref PubMed Scopus (31) Google Scholar) revealed the identical pattern of BMZ staining reported earlier (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar). Western blot analysis using these COOH-Abs was consistent except in one respect. Only the original type XV COOH-Ab, prepared from the early bleeds of one rabbit, reacted with a 116-kDa collagenase-sensitive protein 2Because later bleeds from this rabbit did not detect the 116-kDa protein and supplies of earlier bleeds have been exhausted, we have no further information on the origin of this protein. found in extracts from several human tissues (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar). However, all four type XV COOH-Abs identified a 27-kDa collagenase-resistant fragment (Fig. 1,lane 2, and Ref. 9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar), which was the size expected for the 256-residue carboxyl-terminal noncollagenous domain (see Fig.9). In addition, all four COOH-Abs reacted with very high molecular mass, collagenase-sensitive material, which remained in a 5% stacking gel of an 8% separating gel (used in prior SDS-PAGE, Ref. 9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar), but which appeared as a diffuse area ≥400 kDa upon migration through a 3% stacking gel into a 5% separating gel (Fig.1, lane 3).Figure 9Schematic diagram showing major structural features of the mature human type XV collagen chain, the epitopes for the NH2- and COOH-Abs, and the location of the multiple polypeptide forms and bacterial collagenase cleavage fragments.The three domains of type XV collagen are drawn proportional to their size. The amino terminus (diagonal stripes) contains 530 residues, the discontinuous collagenous region (white and gray areas) encompasses 577 residues, and the carboxyl terminus (dotted pattern) includes 256 residues (3.Myers J.C. Kivirikko S. Gordon M.K. Dion A.S. Pihlajaniemi T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10144-10148Crossref PubMed Scopus (65) Google Scholar, 13.Kivirikko S. Heinämäki P. Rehn M. Honkanen N. Myers J.C. Pihlajaniemi T. J. Biol. Chem. 1994; 269: 4773-4779Abstract Full Text PDF PubMed Google Scholar, 14.Muragaki Y. Abe N. Ninomiya Y. Olsen B.R. Ooshima A. J. Biol. Chem. 1994; 269: 4042-4046Abstract Full Text PDF PubMed Google Scholar).White areas in the collagenous domain indicate the presence of continuous G-X-Y triplets; interveninggray areas and dashed linesin the collagenous subdomains lines show the location of large and small interruptions, respectively. Potential O-linked GAG attachment sequences (D/E-X 1–2-S-G/A: eight in the amino terminus and four in the interruptions) are designated byvertical ball and stick symbols. C = cysteines: two in the amino-terminal domain, two in the collagenous region, and four in the carboxyl-terminal domain. The two cysteines in the collagenous region that are predicted to be involved in interchain disulfide bonds are tagged with an asterisk. The heavy black lines, drawn above the type XV map, show the location of amino- and carboxyl-terminal sequences used to prepare the synthetic peptide and recombinant protein antigens for production of the NH2- and COOH-Abs, respectively. The predicted positions of the 250- and 225-kDa forms, and the collagenase-resistant fragments consisting of the 135-kDa amino-terminal domain and the 27- and 8-kDa carboxyl fragments are designated. The internal boundaries of the 135-, 27-, and 8-kDa fragments coincide with the positions of the nearest recognition sites for bacterial collagenase (G-P-Y↓G-P) (48.Peterkofsky B. Methods Enzymol. 1982; 82A: 453-471Crossref Scopus (100) Google Scholar). Note that molecular mass values of the 250-, 225-, and 135-kDa forms have been derived only from polyacrylamide gel electrophoresis of chondroitinase-treated samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To address the questions presented by these different protein forms, a peptide sequence in the type XV amino terminus was targeted for preparation of another polyclonal antibody (see Fig. 9 and “Materials and Methods”). By immunohistochemistry, this purified antibody (NH2-Ab) showed the same BMZ staining as the COOH-Abs (24.Amenta P.S. Briggs K. Xu K. Gamboa E. Jukkola A.F. Li D. Myers J.C. Hum. Pathol. 2000; 31: 359-366Crossref PubMed Scopus (31) Google Scholar). In Western blot analyses of untreated placenta extracts, the NH2-Ab reacted minimally with the high molecular mass collagenase-sensitive form identified with the COOH-Ab. After collagenase treatment, however, there was no evidence of any size cleavage fragment (expected to be ≥50 kDa, see Fig. 9) corresponding to the type XV amino-terminal domain (data not shown). It therefore appeared that purification steps would be required to further characterize the type XV protein. To this end, fresh frozen placenta was pulverized in liquid nitrogen, and the protein extracted in a 7 m urea buffer under non-reducing conditions (see “Materials and Methods”). Following centrifugation, the solubilized protein was bound to Q-Sepharose and eluted stepwise using increasing concentrations of NaCl. As shown in Fig.2 A, Western blot analysis of the various fractions using the collagenase-resistant, 27-kDa carboxyl-terminal fragment as a marker (9.Myers J.C. Dion A.S. Abraham V. Amenta P.S. Cell Tissue Res. 1996; 286: 493-505Crossref PubMed Scopus (85) Google Scholar), showed that all of the type XV collagen bound to the resin and little was removed in subsequent washing steps using the binding buffer. The protein remained strongly bound in 0.5 m NaCl, but completely eluted at 1m NaCl (Fig. 2 A, lanes 6 and7). The type XV collagen-containing protein pool was concentrated by ultrafiltration and applied to a Sephacryl S-500 gel filtration column under denaturing and disulfide bond-reducing conditions. Dot-blot screening of all fractions showed that the α1(XV) chain eluted in a broad peak with an apparent mass of 300–400 kDa (data not shown). The above concentrated pool, examined by Western blotting using the COOH-Ab, showed the expected 27-kDa collagenase-resistant fragment after digestion (Fig. 2 B, lane 2). Now, for the first time, a strong immunoreactive signal was seen us" @default.
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• W2018096448 date "2000-07-01" @default.
• W2018096448 modified "2023-10-16" @default.
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