Identification of redundant angiogenic sites in laminin α1 and γ1 chains

Abstract

The degradation of the extracellular matrix is one of the first steps involved in angiogenesis, the formation of new vessels from preexisting ones. Laminin, a large extracellular matrix protein, has many biological activities, including the promotion of angiogenesis. Screening of the laminin-1 chains identified 20 angiogenic peptides, of which, A13 and C16, from the α1 and γ1 chains, respectively, were the most active. We recently identified the receptors for C16 as the integrins α5β1 and αvβ3. Here, we show unexpectedly that A13 is a redundant active site to C16 present in the N-terminal globular domain of the α1 chain. The peptides are located in homologous sites present in the last globular domains of their respective chains, and their amino acids are 66% conserved, as compared to the inactive homologous site in the β1 chain, B19 to B20, which is only 18%–23% conserved. Cell attachment studies demonstrated that both A13 and C16 reciprocally inhibited their adhesion activity, whereas the corresponding laminin β1 chain peptides were inactive. Chorioallantoic membrane assays showed that the in vivo angiogenic activity of A13 is blocked by a C16 antagonist, C16S, which also binds to the same integrin receptors. A13 affinity chromatography and immunoprecipitation analysis showed that the αvβ3 and α5β1 integrin receptors bind to this sequence. We have therefore identified redundant activity on two laminin chains. These highly conserved functional sites are likely important mediators of the biological responses of laminins because either one or both of these chains (active sites) are present in almost all laminin isoforms identified to date.

Introduction

The first step in angiogenesis involves breakdown of the basement membrane matrix that allows endothelial cells to migrate, sprout, and form new vessels. The basement membrane contains many bioactive molecules that affect endothelial cell behavior when released during its degradation, including growth factors (i.e., FGFs, PDGF, TGFβ) and extracellular matrix components, such as laminin, a major basement membrane protein [1], [2], [3]. Thus, it is conceivable that as a result of basement membrane degradation, additional angiogenic stimuli are released and become available to sustain new vessel formation [4].

A variety of angiogenic stimulators and inhibitors have been identified which are small bioactive molecules contained within larger molecules. These small molecules are generated during proteolysis or protein processing and include thymosin α1, collagen IV fragments (NCI domains), vasculostatin, endostatin, and angiostatin [5], [6], [7], [8], [9], [10]. Laminin is highly protease sensitive, and it is likely that many small fragments are generated during the breakdown of the basement membrane matrix [11]. Laminin is a high molecular weight (M_r = 900,000) glycoprotein composed of three chains: α, β, and γ. Five different α, three β, and three γ chains, which assemble into at least 12 different laminin isoforms, have been identified to date [12], [13], [14]. We recently screened laminin-1 (α1β1γ1) for angiogenic activity using 12-mer overlapping synthetic peptides to all three chains. Twenty angiogenic peptides were identified which promoted endothelial cell adhesion, migration, and sprouting from aortic rings [15], [16]. The most highly active peptides were identified and included peptide A13 (residues 121 to 133 on the α1 chain), B160 (residues 1607 to 1618 on the β1 chain), and C16 (residues 139 to 149 on the γ1 chain). C16 was very angiogenic in vivo and bound to integrins α5β1 and αvβ3 [17]. Its effect was not inhibited by an RGD peptide and did not involve MAP kinase [17]. Here, we have investigated the most active peptide on the α1 chain, A13. Our data show that A13 is a homologous site on the α1 chain to C16 on the γ1 chain, has considerable sequence homology to C16, competes for adhesion to itself, as expected, and to C16, and utilizes the same cell surface receptors. The homologous site on the β1 chain was not active and demonstrated less sequence homology. These unexpected findings of redundant active sites on laminin indicate a conservation of activity and suggest the importance of this domain as a functional site on laminin-1.