Cloning and Complete Primary Structure of the Mouse Laminin α3 Chain

Abstract

We have isolated and characterized overlapping cDNA clones encoding the ␣3A and ␣3B chains of mouse laminin 5. Sequence analysis of the cDNA for the ␣3B predicts a polypeptide of 2541 amino acids (279,510 Da) comprising a truncated short arm and a carboxyl-terminal long arm common to the laminin ␣ chains identified thus far. The short arm of the ␣3B chain harbors two alternating epidermal growth factor-like domains and two globular domains. The amino-terminal globular domain, thought to mediate interactions with molecules of the extracellular matrix, shows no significant homology to any globular domain at the tips of the known laminin isoforms. The ␣3A cDNA predicts a polypeptide of 1711 amino acids (186,230 Da) that substitutes a short sequence of 43 amino acids for the short arm seen in the ␣3B isoform and displays 77% conservative homology to the ␣3Ep chains of the adhesion ligand epiligrin. Northern and Western blot analyses of skin and lung epithelial cells demonstrated the tissue-specific expression of the laminin ␣3A and ␣3B isoforms, and in situ hybridization on mouse embryos revealed a focal localization of ␣3B in areas of the central nervous system.

Murine Laminin α3A and α3B Isoform Chains Are Generated by Usage of Two Promoters and Alternative Splicing

“…2). Analysis of the nucleotide sequences revealed a TATA box 27 bp upstream to the transcription start site defined previously (11). No potential splice acceptor sites could be identified, suggesting that this DNA fragment is not an intron.…”

“…The resulting constructs were transfected into PAM 212 mouse keratinocytes Nucleotide numbering is relative to the first nucleotide of the codon for the initiation methionine (ATG). Asterisks represent the location of the 5Ј end of the ␣3A cDNA described previously (11). Position and polarity of the oligonucleotides used to amplify the 1-kb fragment are indicated by arrows.…”

“…Genomic Library Screening-The genomic library was screened by plaque hybridization with the ␣-32 P-labeled cDNA probes PR6 and MR10 encoding the 5Ј domains of laminin ␣3A and ␣3B chains, respectively (11). Two nonoverlapping phages, A (15 kb) and B (20 kb), were isolated and further analyzed.…”

“…Recently, we have isolated and characterized the cDNA encoding the ␣ chain of the mouse laminin-5, and we have identified two distinct isoforms ␣3A and ␣3B which differ in their amino-terminal domains (11). The ␣3B polypeptide bears two globular domains and an epidermal growth factor-like domain that substitute the short amino-terminal domain of the ␣3A isoform.…”

“…Laminin-5 (␣3␤3␥2) is a unique laminin variant substantially truncated within the short arm domains compared with the other laminin chains (7)(8)(9)(10)(11). Laminin-5 is secreted into the basement membrane by the basal cells of stratified and transitional epithelia with predominant secretory or protective functions (12).…”