Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

Abstract

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.

Metal Binding Is Critical for the Folding and Function of Laminin Binding Protein, Lmb of Streptococcus agalactiae

“…Dot blot analysis has been carried out to study the interaction of Lmb with EHS laminin, another laminin variant, from mouse sarcoma. EHS and HP laminins are isotypes which share nearly the same structural organization [2]. Dot blot analysis confirms the affinity of Lmb to both types of laminin (Fig.…”

“…Its colonization and invasion into the blood stream is mediated by bacterial surface adhesins, their preferred targets being human extracellular matrix proteins like laminin, fibrinogen, collagen and fibronectin. Laminin is a 900 kDa multidomain glycoprotein of the extracellular matrix and forms a major component of the basement membrane [2].…”

“…This indicates that Lmb and Lbp, being co-transcribed with a zinc binding protein, are likely to play an important role in zinc homeostasis. Interestingly, among the SBPs characterized so far, only the streptococcal proteins PsaA [13] and Lbp [14] are known to function as adhesins as in the case of Lmb [2]. Studying the dual role played by Lmb and its homologs in adhesion and metal homeostasis can therefore provide profound insights into the function of these proteins.…”

Characterization of native laminin from bovine kidney and comparison with other laminin variants

“…Antisera against mouse Ble and B2e (Brown et al, 1990) stained their respective antigens which were present in all laminin preparations (Fig. 4C, D).…”

“…Antisera against laminin chains Ble and B2e (Brown et al 1990) were a kind gift from Dr Peter Taylor (Ciba-Geigy). A monoclonal antibody (C4; Hunter et al, 1989) against laminin Bls was from the Developmental Studies Hybridoma Bank, University of Iowa.…”