Identification of an Active Site on the Laminin α5 Chain Globular Domain That Binds to CD44 and Inhibits Malignancy

Abstract

The laminin ␣5 chain is a component of laminin-10 (␣5␤1␥1) and -11 (␣5␤2␥1). In this study, we have screened 113 overlapping synthetic peptides from the laminin ␣5 globular domain (G-domain) for cell attachment activity with B16-F10 cells using peptide-coated dishes. Eleven attachment-active peptides were identified. In vivo experimental B16-F10 pulmonary metastasis and primary tumor growth assays found that 4 of the 11 peptides inhibited tumor metastasis and growth and increased apoptosis. These four peptides also blocked tumor cell migration, invasion, and angiogenesis. Two of the peptides were highly homologous and showed significant similarity to sequences in collagens. We sought to identify the B16-F10 cell surface receptors for each of the four active peptides using peptide affinity chromatography. Only one peptide recognized a cell surface protein. Peptide A5G27 (RLVSYNGIIFFLK, residues 2892-2904) bound a diffuse M r ϳ120,000 -180,000 band that eluted with 2 M NaCl. Glycosidase digestion of the 2 M eluate yielded protein bands of M r 90,000 and 60,000 that reacted in Western blot analysis with antibodies to CD44. Immunoprecipitation of the A5G27-bound membrane proteins with various cell surface proteoglycan antibodies confirmed CD44 as the surface receptor for A5G27. Finally, attachment assays to A5G27 in the presence of soluble glycosaminoglycans (GAGs) identified the GAGs of CD44 as the binding sites for A5G27. Our results suggest that A5G27 binds to the CD44 receptor of B16-F10 melanoma cells via the GAGs on CD44 and, thus, inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner.

Extracellular Matrix Microenvironment in Glioma Progression

“…Laminins, which were found in blood vessels and in the glial limitants externa in glioma, were also shown to be expressed by human glioma cells positive for glial fibrillary astrocytic protein (GFAP) (Tysnes et al 1999). An active site on laminin which was capable of binding to CD44 was identified (Hibino et al 2004). In addition, Ljubimova et al (Ljubimova et al 2004) found that highly invasive GBMs overexpressed laminin-8, a member of the subset of laminins characterized by containing the alpha4 chain.…”

A novel antiapoptotic mechanism based on interference of Fas signaling by CD44 variant isoforms

“…7 In melanoma cells, CD44 can bind the laminin a5 chain which results in inhibition of tumor cell migration, invasion and angiogenesis. 8 While the standard form of CD44 (CD44s) is ubiquitously expressed, the CD44v isoforms are highly restricted to processes like leukocyte activation, inflammation and malignant transformation (as reviewed by Herrlich et al 9 and Wittig et al 10 ).…”

Laminin α4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

“…Studies of LN␣4-deficient mutant mice (lama4Ϫ/Ϫ) reveal that although ␣4-LNs are not required for blood vessel formation, they play important roles in blood vessel maturation, and in stabilization of vessels that form with injury, inflammation and tumor growth. 7,12,13 In vitro studies indicate that ␣4LNs directly regulate endothelial cell proliferation and inhibit apoptosis.…”