Characterization of Laminin 5B and NH2-terminal Proteolytic Fragment of Its α3B Chain

Abstract

Various laminin isoforms have specific biological functions depending on their structures. Laminin 5A, which consists of the three truncated chains ␣3A, ␤3, and ␥2, is known to have strong activity to promote cell adhesion and migration, whereas a laminin 5 variant consisting of a full-sized ␣3 chain (␣3〉) and the ␤3 and ␥2 chains, laminin 5B, has not been characterized yet. In the present study, we for the first time cloned a fulllength human laminin ␣3B cDNA and isolated the human laminin 5B protein. The molecular size of the mature ␣3B chain (335 kDa) was approximately twice as large as the mature ␣3A chain in laminin 5A. Laminin 5B had significantly higher cell adhesion and cell migration activities than laminin 5A. In addition, laminin 5B potently stimulated cell proliferation when added into the culture medium directly. Furthermore, we found that the ␣3B chain undergoes proteolytic cleavage releasing a 190-kDa NH 2 -terminal fragment. The 190-kDa fragment had activities to promote cellular adhesion, migration, and proliferation through its interaction with integrin ␣ 3 ␤ 1 . These activities of the NH 2 -terminal structure of the ␣3B chain seem to contribute to the prominent biological activities and the physiological functions of laminin 5B.Laminins are an important family of extracellular matrix proteins, which are mainly localized in the basement membranes of various tissues and regulate various cellular functions including cell adhesion and migration (1, 2). The different combinations of five ␣, three ␤, and three ␥ chains give rise to at least 15 laminin heterotrimers with the well known crossshaped structure (1). Of the three laminin subunits, the ␣ chain is most important in determining the tissue-specific distribution and specific biological activity of the laminin isoforms (3). The laminin ␣ chains are classified into two groups, "full-sized" (␣1, ␣2, ␣3〉, and ␣5) and "truncated" (␣3A and ␣4) (1). The amino-terminal region of the full-sized ␣ chains contains three globular domains (VI, IVb, and IVa) and three rodlike domains (V, IIIb, and IIIa) consisting of laminin type epidermal growth factor repeats. The laminin NH 2 -terminal domain VI, which is completely absent in the truncated ␣ chains (␣3A and ␣4), is thought to be essential for the self-assembly and co-polymerization of laminins into large noncovalent networks in the basement membranes and have several cell-binding sites (1). On the other hand, all ␣ chains contain a globular (G) domain consisting of five laminin G modules (G1 to G5) at the COOH terminus. The COOH-terminal G domain of the ␣ chains plays major roles in the interaction with cell surface receptors such as integrins, syndecans, and dystroglycan (4 -6).Typical laminins are produced by the assembly of different ␣ chains with the laminin ␤1 or ␤2 and ␥1 chains, whereas laminin 5 (LN5) 1 is produced by the combination of ␣3A, ␤3, and ␥2 chains. The ␤3 and ␥2 chains are found only in LN5, and all the three LN5 subunits are truncated in the short arms (NH 2 -terminal regions). Cons...

Laminin-3B11, a Novel Vascular-type Laminin Capable of Inducing Prominent Lamellipodial Protrusions in Microvascular Endothelial Cells

“…Cell Adhesion Assay-Cell adhesion assay was performed as described previously (15). Briefly, each well of the 96-well enzyme-linked immunosorbent assay plates (Costar, Cambridge, MA) was coated with various concentrations of laminins at 4°C overnight and then blocked with 1% (w/v) BSA.…”

“…Human recombinant Lm3B32 was purified as described previously (15). Mouse monoclonal antibodies against the N-terminal regions of human laminin ␣3A chain (LS␣3c3) and laminin ␣3B chain (F7) have been prepared before (15). A monoclonal antibody against human laminin ␤1 chain (LT-3) was purchased from Chemicon, and one against human laminin ␥1 chain (number 22) was from BD Bioscience.…”

“…Construction and Transfection of Lm3B11 Expression VectorsThe expression vector of human laminin ␣3B chain has been prepared previously (15). Human laminin ␤1 (5361 bp)-and ␥1 (4830 bp)-chain cDNAs were constructed as follows.…”

“…Mouse EHS laminin-111 (Lm111) and fibronectin were purchased from Chemicon (Temecula, CA). Human recombinant Lm3B32 was purified as described previously (15). Mouse monoclonal antibodies against the N-terminal regions of human laminin ␣3A chain (LS␣3c3) and laminin ␣3B chain (F7) have been prepared before (15).…”

“…We previously expressed laminin-3B32 (Lm3B32; ␣3B/␤3/␥2) as a recombinant protein and revealed its biological activity (15). In addition, our recent immunohistochemical study showed that the ␣3B chain is colocalized with the ␤3 and ␥2 chains in the epithelial BMs of the skin, esophagus, breast, and lung, whereas in the BMs of blood vessels it is colocalized with the ␤1/2 and ␥1 chains, suggesting the presence of novel vascular laminin isoforms, laminin-3B11 (Lm3B11) and laminin-3B21 (Lm3B21) in the vascular BMs (16).…”