Jeanne C. Myers

Type XV collagen exhibits a widespread distribution in human tissues but a distinct localization in basement membrane zones.

Abstract.The collagen family of proteins consists of 19 types encoded by 33 genes. One of the more recently discovered collagens is the α1 chain of type XV. Type XV collagen is comprised of a 577-amino-acid, highly interrupted, triple-helical region that is flanked by amino and carboxy noncollagenous domains of 555 and 256 residues, respectively. To address questions of where this collagen is localized and what its function may entail, we produced a bacteria-expressed recombinant protein representing the first half of the type XV collagen carboxy-terminal domain in order to generate highly specific polyclonal antisera. Immunoscreening of an expression library with the affinity-purified antibody revealed three clones coding for part of the type XV triple-helical region and the entire noncollagenous carboxy-terminus. Western blot analysis of human tissue homogenates identified a 116-kDa collagenase-sensitive protein and a 27-kDa collagenase-resistant fragment, whose electrophoretic mobilities were unchanged in the presence and absence of reductant. Northern blot hybridization to human tissue RNAs indicated that type XV has a prevalent and widespread distribution. To determine the precise localization of type XV collagen, immunohistochemical analyses at the light- and electron-microscopic levels were performed. Type XV exhibited a surprisingly restricted and uniform presence in many human tissues as evidenced by a strong association with vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. These data suggest that type XV collagen may function in some manner to adhere basement membrane to the underlying connective tissue stroma.

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

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.

COOH-terminal propeptides of the major human procollagens. Structural, functional and genetic comparisons.

The sequences of the carboxy-terminal extensions (COOH-propeptides) of at least one chain of all of the major human procollagens have only recently been deduced, and include those of the interstitial (alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III)), basement membrane (alpha 1(IV)) and pericellular (alpha 2(V)) procollagens. Comparisons of DNA and protein sequences, corresponding to these COOH-propeptides domains, established the early divergence of the basement membrane alpha 1(IV) COOH-propeptide from the corresponding sequences of the interstitial and pericellular procollagens. The latter are relatively highly conserved and share 58% primary peptide sequence similarities, whereas sequence similarities relative to alpha 1(IV) are limited. Hydropathy profiles and secondary structure potentials further emphasize the clustering of conserved and variable regions among the interstitial and pericellular COOH-propeptides, and provided further evidence for significant structural differences between these sequences and the alpha 1(IV) COOH-propeptide. The most highly conserved sequences of the alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III) and alpha 2(V) COOH-propeptides include regions surrounding the carbohydrate attachment site, cysteine-containing regions and the COOH-terminal sequences. Cysteinyl, tyrosyl and tryptophanyl residues were found to be highly conserved as were most charged residues. Localization of variable regions, in general, occurs within hydrophilic sequences with high beta-turn potentials that are proximal to intron/exon splice junctions. The most variable sequences are associated with the telopeptides and adjoining NH2-terminal portions of the COOH-propeptides as demonstrated by predictive secondary structure analyses. These results, combined with similar analyses of abnormal alpha 2(I) COOH-propeptide (osteogenesis imperfecta) permitted the identification of subsequences that are likely to be a prerequisite for COOH-propeptide functions, namely procollagen chain recognition and nucleation sites for triple helix formation. These functions are also common to the alpha 1(IV) COOH-propeptide; however, the lack of cleavage of this region and its additional postulated structural role in extracellular matrix interactions likely account for its divergent primary and secondary structure.

Proteoglycan-Collagen XV in Human Tissues Is Seen Linking Banded Collagen Fibers Subjacent to the Basement Membrane

Type 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.

Loss of types XV and XIX collagen precedes basement membrane invasion in ductal carcinoma of the female breast

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. Copyright

Human cDNA clones transcribed from an unusually high-molecular-weight RNA encode a new collagen chain

Human collagen (COL) cDNA clones were isolated from a library representing transcripts synthesized by an established rhabdomyosarcoma (RH) cell line. The 0.6-kb insert of the first isolate encodes a discontinuous collagenous sequence not homologous to type I-XVI COL chains. Sequencing of a second clone with a 4-kb insert revealed an open reading frame (ORF) of 2154 nucleotides. The deduced amino acid (aa) sequence begins with an 186-aa noncollagenous region containing seven cysteines (Cys). Several of the Cys and surrounding aa residues can be aligned with those in type XVI, XII and IX COL. Due to the presence of two long interruptions, the 524-aa collagenous region is separated into three subdomains that each have smaller interruptions of 1-6 aa. The protein terminates with an 8-aa noncollagenous peptide including an unusual single Cys which would be expected to form an interchain disulfide bond. Results of Northern blot hybridization suggest that the new COL chain may be uncommonly large since the clone identified a low-abundance RNA at least 12.4 kb in size. The gene coding for RH COL is located on human chromosome 6. It is now important to elucidate the role of this unusual COL in the infrastructure of extracellular matrix.

Alport syndrome caused by a 5′ deletion within the COL4A5 gene

SummaryFourteen Italian patients affected with X-linked Alport syndrome were analyzed by Southern blotting, using cDNA probes of the COL4A5 gene. One proband was shown to carry a large deletion (> 38 kb) that included the 5′ part of the gene.

Chromosomal assignment of a gene encoding a new collagen type (COL15A1) to 9q21 --> q22.

The collagens constitute a large family of extracellular matrix components primarily responsible for maintaining the structure and biological integrity of connective tissue. These proteins exhibit considerable diversity size, sequence, tissue distribution, and molecular composition. Fourteen types of homo- and/or heterotrimeric molecules, thus far reported, are encoded by a minimum of 27 genes. Nineteen of these genes, including several that are closely linked, have been assigned to 10 separate autosomes, and one collagen gene has been mapped to the X chromosome. We have isolated a 2.1-kb human cDNA clone coding for a collagen molecule different in sequence and structure from types I-XIV collagens. This polypeptide has been designated the alpha 1 chain of type XV collagen. To determine the location of the corresponding gene, the cDNA clone was hybridized to rodent-human hybrid DNAs and to human metaphase chromosomes. The results obtained using the hybrid cell lines showed that this newly identified collagen gene, COL15A1, is present in the pter --> q34 region of chromosome 9. In situ hybridization allowed sublocalization to 9q21 --> q22, a region to which no other collagen genes had previously been assigned. Our data further demonstrate the complex arrangement of the many collagen genes in the human genome.

Mutations in Osteogenesis Imperfecta Leading to the Synthesis of Abnormal Type I Procollagens

Osteogenesis imperfecta or “brittle bone disease” is of interest for several reasons. From the point of view of the physician or of medicine in general, it is of great interest to establish the molecular basis of this disease, or more correctly, group of diseases (for reviews see refs. 1-5). At the phenotypic level, the diseases show a broad spectrum of heterogeneity. Brittle bones are the hallmark of the disease and in the most severe forms, bones are so brittle that death occurs in utero. In the mildest forms, individuals who avoid trauma have minimal disability. Also, in some individuals the disease involves the teeth, skin, and sclerae of the eyes. In others it is confined to the major bony structures. It would obviously be extremely helpful in dealing with patients with these conditions to understand the molecular basis of the diseases and thereby to explain the large heterogeneity in the clinical manifestations. In addition, defining the molecular defects should make it possible to develop DNA tests for the prenatal diagnosis of the severe forms of osteogenesis imperfecta. Hence, at a practical level, it should be possible to diminish greatly the number of new cases. From the point of view of general biology, osteogenesis imperfecta is of interest because it presents a series of highly informative “experiments of Nature.” It is now established that many forms of osteogenesis imperfecta are produced by mutations in the genes for type I procollagen (see refs. 1, 3, and 5). The specific kinds of mutations that are found indicate directly the special vulnerabilities of the genes to mutations. Because the amino acid sequence of the protein is highly repetitive, the coding sequences of the genes are highly repetitive. Experiments in bacteria and other organisms demonstrate that repetitive DNA sequences are prone to undergo extensive recombination during either meiosis or mitosis. Therefore the mutations found in collagen

Type XV collagen in human colonic adenocarcinomas has a different distribution than other basement membrane zone proteins.

In situ carcinomas must penetrate their own basement membrane to be classified as invasive, and subsequently infiltrate surrounding connective tissue and cross vascular basement membranes to metastasize hematogenously. Accordingly, in many studies, integral basement membrane components, including type IV collagen, laminin, and heparan sulfate proteoglycan, have been localized in a spectrum of tumors to gain insight into their role in neoplasia. A number of recently identified extracellular matrix molecules and isoforms of the aforementioned proteins have been localized to the basement membrane zone, illustrating another level of biochemical heterogeneity in these structures. As the complexity of these matrices becomes more apparent, their roles in maintaining homeostasis and in tumor biology falls into question. Of the new group of collagens localized to the basement membrane zone, type XV was the first to be characterized (Cell Tissue Res, 286:493-505, 1996). This nonfibrillar collagen has a nearly ubiquitous distribution in normal human tissues via a strong association with basement membrane zones, suggesting that it functions to adhere basement membrane to the underlying stroma. To begin investigation of this protein in malignant tumors, we have localized type XV in human colonic adenocarcinomas and compared its distribution with that of type IV collagen and laminin. Collagens XV and IV and laminin were found in all normal and colonic epithelial, muscle, fat, neural, and vascular basement membrane zones, as shown previously. In moderately differentiated, invasive adenocarcinomas, laminin and type IV collagen were sometimes observed as continuous, linear deposits around some of the malignant glands, but more often they were seen in either discontinuous deposits or were completely absent. In contrast, type XV collagen was characterized as virtually absent from the basement membrane zones of malignant glandular elements in moderately differentiated tumors. Nevertheless there were also similarities; all 3 proteins were usually present in the stroma and adjacent vascular basement membrane zones surrounding invasive glands. The loss of type XV collagen from these malignant epithelial basement membrane zones and its increased interstitial expression suggests a role for this protein in the invasive process and the possibility that it may provide a sensitive indicator of tumor invasion.