The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase.

Abstract

We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of /3-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frameshift-generated sequence unique to the alternatively spliced form of human /3-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human fl-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized. (J.

Signaling Pathways Transduced through the Elastin Receptor Facilitate Proliferation of Arterial Smooth Muscle Cells

“…Total DNA was assayed at each end point using the DNeasy tissue system. To determine whether the mitogenic signal of -elastin was transmitted through the EBP, quadruplicate cultures of CA SMC were preincubated for 3 h with 50 mM lactose, which causes release of the EBP from the cell surface (1,18,(25)(26)(27)(28)(29), or with 5 g/ml anti-S-GAL antibody, which blocks its elastin binding domain (33). They were then incubated with 2 Ci/ml [ 3 H]thymidine for the next 48 h, in the presence or absence of these reagents and 50 g/ml -elastin as described above.…”

“…Immunoprecipitation-Lysates of control and -elastin-treated cells were also immunoprecipitated with 3 g/ml polyclonal anti-FAK antibody and subsequently treated with 50 l of 4% Protein A-beaded agarose as previously described (33). Immunoprecipitated proteins were then resolved at 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with 2.5 g/ml monoclonal PY20 antibody recognizing phosphotyrosine.…”

“…We have learned, however, that binding of these carbohydrate-bearing moieties causes conformational changes of the 67-kDa protein, so that it loses its ability to bind elastin and separates from the complex with other receptor subunits (1,25). Sequencing of the EBP isolated from arterial SMC and molecular cloning led us to the discovery that this protein is identical to the 67-kDa, enzymatically inactive, alternatively spliced variant of human ␤-galactosidase (S-GAL) and that it binds elastin and laminin through the domain located in the unique frameshift-generated sequence (33,34). We also characterized the 61-kDa subunit of the elastin receptor as sialidase (neuraminidase) (EC 3.2.1.18) and the 55-kDa subunit as the protective protein, also called carboxypeptidase A or cathepsin A (EC 3.4.16.1) (35).…”

Interaction between the Elastin Peptide VGVAPG and Human Elastin Binding Protein

“…It is a heterotrimer comprising a peripheral elastin binding protein (EBP) 5 and two membrane-associated proteins, neuraminidase-1 (EC 3.2.1.18) and the protective protein/cathepsin A (EC 3.4.16.1).…”

“…Although neuraminidase-1 and protective protein/cathepsin A are identical to the subunits found in the lysosome, EBP is an enzymatically inactive spliced variant of lysosomal ␤-gal (5,7,8). The alternative splicing of ␤-gal primary transcripts leading to EBP consists of two deletions.…”