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 (...

S U M M A R YType XV is a large collagen-proteoglycan found in all human tissues examined. By light microscopy it was localized to most epithelial and all nerve, muscle, fat and endothelial basement membrane zones except for the glomerular capillaries or hepatic/ splenic sinusoids. This widespread distribution suggested that type XV may be a discrete structural component that acts to adhere basement membrane to the underlying connective tissue. To address these issues, immunogold ultrastructural analysis of type XV collagen in human kidney, placenta, and colon was conducted. Surprisingly, type XV was found almost exclusively associated with the fibrillar collagen network in very close proximity to the basement membrane. Type XV exhibited a focal appearance directly on the surface of, or extending from, the fibers in a linear or clustered array. The most common single arrangement was a bridge of type XV gold particles linking thick-banded fibers. The function of type XV in this restricted microenvironment is expected to have an intrinsic dependence upon its modification with glycosaminoglycan chains. Present biochemical characterization showed that the type XV core protein in vivo carries chains of chondroitin/dermatan sulfate alone, or chondroitin/dermatan sulfate together with heparan sulfate in a differential ratio. Thus, type XV collagen may serve as a structural organizer to maintain a porous meshwork subjacent to the basement membrane, and in this domain may play a key role in signal transduction pathways. (J Histochem Cytochem 53: [165][166][167][168][169][170][171][172][173][174][175][176] 2005)

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Ductal and lobular carcinomas comprise most malignancies of the female breast and the morbidity and mortality associated with breast cancer. During the progression from in situ to invasive stages, tumour cells penetrate the epithelial and vascular basement membranes (BM) to realize full metastatic potential. While the definition of these structures has primarily resulted from analysis of laminin and type IV collagen, characterization of newly discovered BM/BM zone (BMZ) proteins will further elucidate the interactions between tumour cells and the host stroma. We have studied the expression of two non-fibrillar BMZ collagens, the type XV proteoglycan and collagen XIX, in breast cancer where a linear, well-formed BM becomes fragmented and even lost in the progression of epithelial malignancy. In the normal breast, types XV and XIX were found in all BMZ: epithelial, muscle, neural, endothelial, and fat. In in situ lesions, these two collagens, and particularly type XV, were often absent from the BM/BMZ displaying a continuous or just focally disrupted type IV/laminin staining pattern. In contrast, infiltrating ductal carcinomas showed only rare traces of laminin and collagen IV reactivity adjacent to the glands and tumour nests, and similarly there was little if any evidence of types XV and XIX collagen. All four molecules were, however, detected in the interstitium associated with some of the invasive carcinomas. The data suggest that types XV and XIX collagen are lost early in the development of invasive tumours, prior to penetration and eventual dissolution of the epithelial BM. Disappearance of these proteins from the BM/BMZ may signal remodelling of the extracellular matrix to promote tumour cell infiltration.

Type XIX collagen was discovered from the sequence of rhabdomyosarcoma cDNA clones. The chain is composed of a 268-residue amino terminus, an 832-residue discontinuous collagenous region, and a 19-residue carboxyl peptide. Light microscopy immunohistochemistry of adult human tissues demonstrated that type XIX is localized in vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. It also appears to be involved in events linked to skeletal myogenesis. In this report, we have presented the first direct evidence for the molecular structure of type XIX collagen. Using human umbilical cord, native type XIX was purified by neutral salt extraction and by ion exchange and antibody affinity chromatography. Type XIX was found to represent only ϳ10 ؊6 % of the dry weight of tissue, making it by far the least abundant collagen ever isolated. Transmission electron microscopy after rotary shadowing revealed the appearance of rodlike structures with multiple sharp bends, a small nodule at one end of the molecule, and a total length of 240 nm. Domain-specific antibodies were used to identify the nodule as the noncollagenous amino terminus, whereas the location of most kinks corresponds to major interruptions separating the five collagenous subdomains. More than half of the type XIX molecules observed were present in oligomers of different size and complexity, resulting from association of the amino-terminal domains. Biochemical analysis demonstrated that these supramolecular aggregates are dependent upon and/or stabilized by intermolecular disulfide cross-links and that the globular amino terminus contains a high affinity, heparin-binding site. The polymorphic conformational states of this rare collagen, and its ability to self-assemble into a higher order structure provide focal points for future determination of biologically significant functions in cell-cell and/or cell-matrix interactions.Twenty-six collagen types have currently been designated; seven of these are recent discoveries, and several have yet to be described (1-7). With the diversity that this family has presented, one of the few defining elements is the existence of one or more triple-helical regions, regardless of size and relative proportion to the entire protein. In the most general sense, therefore, collagens have been divided into the classic fibrillar group (i.e. those containing the ϳ333 continuous Gly-X-Y triplets and involved in the formation of cross-striated fibrils) and the nonfibrillar group, a highly heterogeneous class exhibiting a spectrum of sizes, supramolecular assemblies, and chain organization, with the one commonality being the presence of noncollagenous sequences interrupting and/or flanking collagenous domains (1, 2, 8 -10). Understanding the complex structure and function of these many proteins has proven to be a formidable task despite, in many instances, extensive knowledge of disease phenotypes directly attributable to the respective collagen gene mutations (1, 11, 12). A major complication in this process has b...