Marie-France Champliaud

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.

Presence of Laminin α5 Chain and Lack of Laminin α1 Chain during Human Muscle Development and in Muscular Dystrophies

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin α1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin α1 chain. Nevertheless, there seems to be no expression of laminin α1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin α1 chain as a recombinant protein and made antibodies specific for human laminin α1 chain (anti-hLN-α1G4/G5). We also made antibodies to the human laminin α5 chain purified from placenta. In the present report we show that hLN-α1G4/G5 antibodies react with a 400-kDa laminin α1 chain and that 4C7 reacts with a 380-kDa laminin α5 chain. Immunohistochemistry with the hLN-α1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin α5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin α1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.

Collagen XVII and BPAG1 expression in the retina: evidence for an anchoring complex in the central nervous system.

The ectoderm gives rise not only to the skin but also to the entire CNS. This common embryonic lineage suggests that some molecular isoforms might serve analogous functions in both tissues. Indeed, not only are laminins important components of dermal adhesion mechanisms, but they also regulate some aspects of synaptic development in both the CNS and the PNS. In the skin, laminins are part of a hemidesmosome complex essential for basal keratinocyte adhesion that includes collagen XVII (BP180) and BPAG1 (dystonin/BP230). Here, we show that CNS neurons also express collagen XVII and BPAG1 and that these molecules are expressed in the adult and developing retina. In the retina, isoforms of collagen XVII and BPAG1 are colocalized with laminins at photoreceptor synapses and around photoreceptor outer segments; both molecules are expressed by rods, whereas cones express collagen XVII but not BPAG1. Moreover, biochemical data demonstrate that collagen XVII complexes with retinal laminins. We propose that collagen XVII and BPAG1 isoforms may help to anchor elements of the rod photoreceptor cytomatrix to the extracellular matrix. J. Comp. Neurol. 487:190–203, 2005.

Laminins containing the β2 chain modulate the precise organization of CNS synapses

Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic components; however, the precise nature of the intrasynaptic components in the CNS remains obscure. Potential intrasynaptic components include extracellular matrix molecules such as laminins; here, we isolate beta2-containing laminins, including perhaps laminins 13 (alpha3beta2gamma3) and 14 (alpha4beta2gamma3), from CNS synaptosomes suggesting a role for these molecules in synaptic organization. Indeed, hippocampal synapses that form in vivo in the absence of these laminins are malformed at the ultrastructural level and this malformation is replicated in synapses formed in vitro, where laminins are provided largely by the post-synaptic neuron. This recapitulation of the in vivo function of laminins in vitro suggests that the malformations are a direct consequence of the removal of laminins from the synapse. Together, these results support a role for neuronal laminins in the structural integrity of central synapses.

cDNA Cloning and Characterization of Sciellin, a LIM Domain Protein of the Keratinocyte Cornified Envelope

Sciellin is a precursor of the cornified envelopes of mammalian keratinizing tissues. We have cloned the cDNA encoding sciellin by screening a human keratinocyte expression library with a sciellin-specific monoclonal antibody. The composite cDNA of 2.35 kilobase pairs encodes a protein of 75.3 kDa with a pI of 10.09. The translated sequence has a central domain containing 16 repeats of 20 amino acids each that is rich in Gln and Lys residues, which are potential transglutaminase substrates, and a carboxyl domain, which contains a single LIM motif. Sciellin cDNA probes hybridize to bands of 3.4 and 4.4 kilobase pairs on Northern blots of cultured human keratinocyte RNA. The gene was mapped to human chromosome band 13q22 by fluorescence in situ hybridization. Radiation hybrid mapping demonstrated that sciellin is linked to the sequence tagged site marker WI-457 with a logarithm of the odds score of 7.77.In situ hybridization of human foreskin tissue sections demonstrated that sciellin is expressed in the stratum granulosum. Immunofluorescent staining with a polyclonal rabbit antibody made to a recombinant sciellin protein showed peripheral cytoplasmic localization in the upper cell layers of epidermis and in stratified squamous epithelia such as the oral cavity, esophagus, and vagina. Simple and columnar epithelia, with the exception of the amnion, showed no reaction.