Donald R. Gerecke

Bone morphogenetic protein 1 is an extracellular processing enzyme of the laminin 5 gamma 2 chain

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (α3β3γ2) within the α3 and γ2 chains (1). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first α3 chain cleavage (200-l65 kDa α3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa α3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The γ chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the γ2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (2). Recombinant BMP-1 cleaves γ2 in vitro,both within intact laminin 5 and at the predicted site of a recombinant γ2 short arm. α3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin α3 and γ2 chains to be substrates for BMP-1 in vitro. We speculate that γ2 cleavage is required for formation of the laminin 5–6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.

Collagen XXIV, a vertebrate fibrillar collagen with structural features of invertebrate collagens: Selective expression in developing cornea and bone

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar α1(V), α1(XI), and α2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the α1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains α1(V) and α1(XI). However, a short imperfection in the triple helix makes α1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.

Low nidogen affinity of laminin-5 can be attributed to two serine residues in EGF-like motif γ2III4

High affinity nidogen binding of laminin‐1 (chain composition α1β1γ1) has been previously mapped to a single EGF‐like motif γ1III4 of its γ1 chain. Two more isoforms, laminin‐5 (α3β3γ2) and laminin‐7 (α3β2γ1), show low and high binding activity, respectively, indicating that the γ2 chain is of low affinity. This was confirmed by recombinant production of the homologous EGF‐like motif γ2III4 of the γ2 chain, which has a 100,000‐fold lower binding activity than γ1III4. The crucial heptapeptide binding sequence Asn‐lle‐Asp‐Pro‐Asn‐Ala‐Val of γ1III4 is modified in γ2III4 by replacing both the central Asn and Val by Ser. Changing these replacements to Asn and Val by site‐directed mutagenesis enhanced the activity of γ2III4 to a level which was only 5‐fold lower than that of γ1III4. Despite their high sequence identity (77%) motifs γ1III4 and γ2III4 were also shown to differ considerably in immunological epitopes. This indicates distinctly different functions for laminins which differ in the γ chain isoform.

Cloning of the {beta}3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa

Abstract Laminin 5 consists of three polypeptides, α3, β3, and γ2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon—intron organization of the human LAMB3 gene. Characterization of five overlapping λ phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 bp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon—intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes.

The mouse α1(XII) and human α1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.