Human laminin M chain (merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues.

The primary structure of the human laminin M chain was determined from cDNA clones isolated from human placental libraries. The clones covered a total of 6,942 bp, with 49-bp encoding a 5' end untranslated region and 6,893-bp coding for a translated sequence. The complete human laminin M chain contains a 22-residue signal peptide and 3,088 residues of the mature M chain. The M chain has a domain structure similar to that of the human and mouse A chains. The homology between the two human laminin heavy chains is highest in the short arm region and lowest in the long arm helical domain I + II. Northern blot analysis of human fetal tissues showed that the M chain was expressed in most tissues such as cardiac muscle, pancreas, lung, spleen, kidney, adrenal gland, skin, testis, meninges, choroid plexus, and some other regions of the brain, but not in liver, thymus, and bone. In situ hybridization localized the expression of the M chain gene to cells of mesenchymal origin. In contrast, expression of the A chain was observed only in kidney, testis, neuroretina and some region of brain as determined by Northern analyses. Epithelial and endothelial cells were negative for both M and A chain gene transcripts. The gene for the human M chain (LAMM) was localized to chromosome 6q22-->23.

Bruton's tyrosine kinase together with PI 3-kinase are part of Toll-like receptor 2 multiprotein complex and mediate LTA induced Toll-like receptor 2 responses in macrophages.

Lipoteichoic acid (LTA) of Gram-positive bacteria initiates innate immune responses via Toll-like receptor-2 (TLR2), resulting in the activation of intracellular signaling and production of inflammatory cytokines in macrophages. Although Bruton's tyrosine kinase (Btk) is biologically important molecule implicated in immune regulation and recently in TLR signaling its importance for LTA-TLR2 mediated responses has not been evaluated. In this study, we detected Btk in the LTA signaling complex with TLR2 and PI 3-kinase (PI3K). The constitutive interaction of these proteins was mediated via PI3K Src homology (SH3) -domain. Both Btk and PI3K were activated by LTA stimulation and the LTA induced cytokine expression was differentially modulated by these kinases. LTA induced the activation of nuclear factor kappaB (NFkappaB), however, only Btk inhibition affected the LTA induced Ser536 phosphorylation and DNA-binding of NFkappaB. In conclusion, our results demonstrate that Btk and PI3K occupy important roles in TLR2-induced activation of macrophages, resulting in selective regulation of cytokines.

Kidney morphogenesis: cellular and molecular regulation.

Development of an organ is directed by cell and tissue interactions and these also occur during the formation of functional kidney. During vertebrate development inductive signalling between mesenchyme and epithelium controls the organogenesis of all three kinds of kidneys: pronephros, mesonephros and metanephros. In higher animals the metanephros differentiates into the permanent kidney and in this review we will mainly concentrate on its development. Molecular interactions currently known to function during nephrogenesis have primarily been based on the use of knockout techniques. These studies have highlighted the role for transcription factors, signalling molecules, growth factors and their receptors and also for extracellular matrix components in kidney development. Finally in this review we will represent our own model for kidney development according to the knowledge of the genes involved in the development of the functional excretory organ, kidney.

The human laminin beta 2 chain (S-laminin): structure, expression in fetal tissues and chromosomal assignment of the LAMB2 gene.

The sequence of the human laminin beta 2 chain (previously s-laminin) was derived from cloned cDNAs. The complete translation product has 1798 amino acid residues, including a 32-residue signal peptide. The human chain lacks the tripeptide sequence LRE in domain I which is present in the rat polypeptide chain and has been shown to promote motor neuronal cell adhesion. The human gene (LAMB2) was localized to chromosome 3p21 using somatic cell hybrids and fluorescent in situ hybridization analysis. Northern and in situ hybridization analyses from numerous fetal tissues revealed that the beta 2 chain is generally widely expressed. beta 2, but not beta 1, was shown by in situ hybridization to be expressed in fetal brain and renal glomeruli. In fetal skin, beta 2 was expressed both in epidermal and dermal cells, while beta 1 was expressed only in the dermis. Expression of beta 2 in fetal liver was seen in hepatocytes, while no signals were observed for beta 1. In lung, both beta 1 and beta 2 were expressed in alveoli and bronchial smooth muscle cells, whereas only the beta 2 chain was expressed in bronchial epithelial cells. In striated muscle, however, the beta 1 chain, but not beta 2, was expressed. These results indicate different biological roles for the laminin beta 1 and beta 2 chains.

Structure of the human laminin alpha2-chain gene (LAMA2), which is affected in congenital muscular dystrophy.

We have determined the structure and complete exon size pattern of the human laminin alpha2-chain gene (LAMA2), which has been shown to be affected in congenital muscular dystrophy (Helbling-Leclerc, A., Zhang, X., Topaloglu, H., Cruaud, C., Tesson, F., Weissenbach, J., Tomé, F. M. S., Schwartz, K., Fardeau, M., Tryggvason, K., and Guicheney, P. (1995) Nat. Genet. 11, 216-218). The gene is over 260, 000 base pairs and contains 64 exons. The sequence of all exon-intron borders was determined. Two of the exons, i.e. exons 43 and 52, are extremely small in size, 6 and 12 base pairs, respectively. Comparison of the exon pattern of the human LAMA2 gene with that of the Drosophila LAMA gene revealed that only 2 of 63 intron locations in the 5'-end of the human gene match the intron locations in the Drosophila gene, which contains 14 introns.

Structure of the human laminin B1 chain gene.

The exon-intron structure of the human laminin B1 chain gene was determined from genomic clones that spanned 90 kilobase pairs (kb), including 80 kb of the structural gene, about 1 kb of the 5'-flanking region, and 9 kb of the 3'-flanking region. DNA sequencing and heteroduplex analyses demonstrated that the gene consists of 34 exons. The intron sizes vary from 92 base pairs to more than 15,000 base pairs. The clones did not completely contain introns 13 and 14 and, therefore, the exact size of the gene remains to be determined. The first exon encodes a 5'-untranslated region, with the ATG translation start codon being in exon 2. The promoter region does not contain TATA or CAAT boxes, but it has four GC boxes. Additionally, two GC boxes are located in the 5'-untranslated sequence. A glucocorticoid response element-like sequence and a sequence resembling the binding sequence for transcription factor AP-2 are present in the 5'-flanking region. Another potential AP-2 binding sequence is located in the 5'-untranslated sequence of exon 1. A TGACC motif present in the binding region for the retinoic acid response element of the mouse laminin B1 gene promoter is also present in the human counterpart. The overall exon pattern of the gene correlates only slightly with the highly conserved structural domains and internal repeats of the B1 polypeptide chain. Furthermore, the exon profile differs considerably from that of the laminin B2 gene. A HincII/HpaI restriction fragment length polymorphism was identified in exon 31.

Primary structure of the human laminin A chain. Limited expression in human tissues.

cDNA clones for the human laminin A chain were isolated from libraries prepared from human gestational choriocarcinoma cell line (JAR) RNA. They cover approx. 8 kb from the 5'-end of the 9.5 kb mRNA coding for this protein. Our clones contain 94 nucleotide residues for the 5'-end untranslated region and 7885 nucleotide residues of coding sequence. The complete human laminin A chain contains a 17-amino acid-residue signal peptide and a 3058-residue A chain proper. The human laminin A chain has a distinct domain structure with numerous internal cysteine-rich repeats. The large globular domain G has five repeats, which have several conserved glycine and cysteine residues. Furthermore the A chain contains 20 internal cysteine-rich repeats present in tandem arrays in three separate clusters (domains IIIa, IIIb and V). Domain I + II has a predicted continuous alpha-helical structure characterized by heptad repeats and three domains (IVa, IVb and VI) are predicted to contain a number of beta-sheets and coiled-coil structures. Northern-blot analysis was used to study the laminin A chain expression in the JAR cell line, full-term placenta and newborn-human tissues (kidney, spleen, lung, heart muscle, psoas muscle and diaphragm muscle). The expression was detectable in newborn-human kidney and JAR cell line only. The overall amino acid sequence identity between human and mouse is 76%. The human chain has only one Arg-Gly-Asp (RGD) sequence, which is located in the long arm within domain G, whereas the single RGD sequence in the mouse chain is located in the short arm in domain IIIb. The degree of identity between the human laminin A chain sequence and the sequence available for merosin [Ehrig, Leivo, Argraves, Ruoslahti & Engvall (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3264-3268] is about 41% and when conservative substitutions are included the degree of similarity is 54%.