Low nidogen affinity of laminin-5 can be attributed to two serine residues in EGF-like motif gamma 2III4.

High affinity nidogen binding of laminin-1 (chain composition alpha 1 beta 1 gamma 1) has been previously mapped to a single EGF-like motif gamma 1III4 of its gamma 1 chain. Two more isoforms, laminin-5 (alpha 3 beta 3 gamma 2) and laminin-7 (alpha 3 beta 2 gamma 1), show low and high binding activity, respectively, indicating that the gamma 2 chain is of low affinity. This was confirmed by recombinant production of the homologous EGF-like motif gamma 2III4 of the gamma 2 chain, which has a 100,000-fold lower binding activity than gamma 1III4. The crucial heptapeptide binding sequence Asn-Ile-Asp-Pro-Asn-Ala-Val of gamma 1III4 is modified in gamma 2III4 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 gamma 2III4 to a level which was only 5-fold lower than that of gamma 1III4. Despite their high sequence identity (77%) motifs gamma 1III4 and gamma 2III4 were also shown to differ considerably in immunological epitopes. This indicates distinctly different functions for laminins which differ in the gamma chain isoform.

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

Laminin 5 consists of three polypeptides, alpha 3, beta 3, and gamma 2, 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 lambda 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.

A homozygous nonsense mutation in the beta 3 chain gene of laminin 5 (LAMB3) in Herlitz junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (H-JEB) is a severe autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Based on immunofluorescence analysis recognizing laminin 5 epitopes (previously known as nicein/kalinin), the genes for this lamina lucida protein have been proposed as candidate genes in H-JEB. In this study, we examined the gene encoding the beta 3 polypeptide chain of laminin 5 (LAMB3) by Northern hybridization and RT-PCR analysis of keratinocyte mRNA from a proband in a family with H-JEB. Northern analysis revealed markedly reduced levels of the laminin beta 3 chain mRNA. Amplification of mRNA by RT-PCR, followed by direct nucleotide sequencing, revealed a homozygous C-to-T transition resulting in a premature termination codon (CGA --> TGA) on both alleles. This mutation was verified at the genomic DNA level, and both parents were shown to be heterozygous carriers of the same mutation. This is the first description of a mutation in the laminin beta 3 chain gene (LAMB3) of laminin 5 in an H-JEB patient.

The complete primary structure for a novel laminin chain, the laminin B1k chain.

We have isolated overlapping cDNA clones encoding the entire kalinin B1 chain. The predicted sequences are consistent with the models proposed for the structure of this chain as a truncated homologue of the B1 chain of laminin. The percent sequence homology with other laminin B1 chains is relatively low, suggesting that this chain is functionally different. The sequence of the VI domain of this chain is consistent with the possibility that it serves to bind specifically the kalinin A chain and subsequently covalently binds to it. In keeping with the accepted nomenclature for the laminin chains we name this novel polypeptide the laminin B1k chain.

The 100-kDa chain of nicein/kalinin is a laminin B2 chain variant.

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and II) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105-155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (approximately 5200 nucleotides) and amino acid sequence obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that nicein/kalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain III that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since nicein/kalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-dermal adhesion.

Injection of 1,25-(OH)2 vitamin D3 enhances resistance artery contractile properties.

The hypothesis that 1,25-dihydroxyvitamin D3 [1,25-(OH)2 vitamin D3] modulates vascular smooth muscle contractile function was tested. 1,25-(OH)2 vitamin D3 (50 ng/day) was administered by intraperitoneal injection over a 3-day period to 13-15-week-old male spontaneously hypertensive and Wistar-Kyoto normotensive rats. On the fourth day, serum was prepared and contractile force generation of isolated mesenteric resistance arteries was examined. Treatment with 1,25-(OH)2 vitamin D3 approximately doubled serum levels of the hormone and increased ionized and total serum Ca2+ and phosphate by 5-10%. No effect on blood pressure was detected. 1,25-(OH)2 vitamin D3 injection in both strains enhanced maximal stress generation to norepinephrine and serotonin by 30-40%, with no effect on apparent sensitivity of the vessels to the agonists. To assess the effect of a maneuver that elevates serum ionized Ca2+ without the addition of exogenous hormone, maximal stress generation was examined in resistance arteries isolated from rats fed diets containing 0.5% or 2% calcium over a 6-7-week period. Maximal stress generation in response to norepinephrine was greater in vessels from rats of both strains maintained on 0.5% calcium. It is concluded that 72-hour in vivo treatment with 1,25-(OH)2 vitamin D3 increases contractile force-generating capacity of resistance arteries without affecting blood pressure. It is proposed that this action of 1,25-(OH)2 vitamin D3 is the result of a direct action of the hormone on the vascular wall.

Vascular actions of cicletanine.

Vascular wall hypertrophy and enhanced vascular reactivity are characteristic of the hypertensive state. Pharmacologic reversal or prevention of these changes might be of therapeutic benefit. We examined the mechanism by which cicletanine, an antihypertensive vasodilator, induces relaxation of isolated mesenteric resistance arteries and assessed its effects on growth of cultured mesenteric artery myocytes. Initial experiments determined the effect of cicletanine on aortic rings and superior mesenteric arteries isolated from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Cicletanine induced dose-dependent relaxation of pre-contracted segments of both artery types. Aortas of SHR were more sensitive to cicletanine than those of the WKY, while no difference in sensitivity of mesenteric arteries was observed between the two strains. Within-strain comparisons showed that aortas of SHR were more sensitive to cicletanine than mesenteric arteries while no differences were detected in vessels of the WKY. To assess the mechanism of action, the effect of cicletanine on intracellular Ca2+ was determined in mesenteric resistance arteries loaded with fura-2. In 4 of 4 preparations tested, 100 microM cicletanine induced a rapid fall in the level of free ionized Ca2+. When the endothelial lining of superior mesenteric arteries was removed or vessels were pre-treated with either 1 microM propranolol, 0.1 mM ouabain or 5 microM indomethacin, no effect on cicletanine-induced relaxation (10-300 microM) was detected.(ABSTRACT TRUNCATED AT 250 WORDS)