Basement Membrane Zone Type XV Collagen Is a Disulfide-bonded Chondroitin Sulfate Proteoglycan in Human Tissues and Cultured Cells

Abstract

Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The ␣1(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 for O-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 ␣ 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 ␣ 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 ϳ8-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 ϳ19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and Stä ndker, L. (1999) Biochemistry 38, 10217-10224), which may be liberated from the ␣1(XV) chain. 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). 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), 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 (3-6). 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 (...

Complete Suppression of Tumor Formation by High Levels of Basement Membrane Collagen

“…This cross-reactant is also seen in the vector-only control clones and in the parental D98 AP2 line (not shown). The detection of two forms of type XV collagen in some cell types (e.g., clone 15:6) was previously reported at the time when the specificity of the antibody was established (13).…”

“…Three vectoronly control clones were also analyzed (DP2, DP3, and DP4). The human type XV collagen protein migrates with a molecular weight of f250 kDa (13) and the antibody to it cross-reacts with another unknown protein of higher molecular weight (which shows no correlation with collagen XV expression). This cross-reactant is also seen in the vector-only control clones and in the parental D98 AP2 line (not shown).…”

Structure and Function of Collagen‐Derived Endostatin Inhibitors of Angiogenesis

“…Collagens XV and XVIII are also known to be proteoglycans being extensively modified by either chondroitin sulfate (XV) or heparan sulfate (XVIII) side chains, respectively (11,12). Potential SGD/SGE attachment sequences for these glycosaminoglycans (GAG) are primarily located in the mucin-like segments, but there are also a few in the noncollagenous interruptions of Figure 1.…”

“…The two attachment sites predicted for collagen XVIII have in fact been shown by recombinant production to be substituted (T. Sasaki, R. Timpl, unpublished). Several more attachment sites exist probably for collagen XV (11). It is also likely that several Thr residues in the mucin-like regions are modified by short O-linked oligosaccharides.…”

Heparan sulfate proteoglycans: heavy hitters in the angiogenesis arena

“…This proteoglycan may carry up to four heparan sulfate chains, each attached to one of the 11 noncollagenous domains (Figure 1). The recent discovery that collagen type XV, the second member of the multiplexin gene family, is also a hybrid proteoglycan carrying chondroitin sulfate chains (9) indicates that the noncollagenous domains of these molecules are highly suitable for glycosaminoglycan substitution.…”