METH-1, a Human Ortholog of ADAMTS-1, and METH-2 Are Members of a New Family of Proteins with Angio-inhibitory Activity

doi:10.1074/jbc.274.33.23349

METH-1, a Human Ortholog of ADAMTS-1, and METH-2 Are Members of a New Family of Proteins with Angio-inhibitory Activity*

^‡Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, California 90095, ^¶Human Genome Sciences, Rockville, Maryland 20850, and ^‖Departments of Obstetrics and Gynecology and Biological Chemistry, Jonsson Cancer Center, UCLA, Los Angeles, California 90095

Abstract

We have studied two related proteins that contain a repeated amino acid motif homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (TSP1). Complete sequence analysis revealed no other similarities with TSP1, but identified unique signal sequences, as well as metalloprotease and disintegrin-like domains in the NH₂ termini. We named these proteins METH-1 and METH-2 due to the novel combination of metalloprotease andthrombospondin domains. Overall amino acid sequence identity between METH-1 and METH-2 is 51.7%, yet transcript distribution revealed non-overlapping patterns of expression in tissues and cultured cell lines. To characterize these proteins functionally, we isolated full-length cDNAs, produced recombinant protein, and generated antisera to the recombinant proteins. Both METH-1 and METH-2 represent single copy genes, which encode secreted and proteolytically processed proteins. METH proteins suppressed fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited vascular endothelial growth factor-induced angiogenesis in the chorioallantoic membrane assay. Suppression of vessel growth in both assays was considerably greater than that mediated by either thrombospondin-1 or endostatin on a molar basis. Consistent with an endothelial specific response, METH-1 and METH-2 were shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth. We propose that METH-1 and METH-2 represent a new family of proteins with metalloprotease, disintegrin, and thrombospondin domains. The distinct distribution of each gene product suggests that each has evolved distinct regulatory mechanisms that potentially allow for fine control of activity during distinct physiological and pathological states.

Footnotes

↵* This work was supported by National Institutes of Health Grants R29CA63356-01 and R01CA77420-01.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) (METH-1) and (METH-2).
↵§ These authors have contributed equally to the manuscript.
↵** To whom correspondence should be addressed: Molecular Biology Inst., UCLA, 5th Fl., Rm. 559, 611 Circle Dr. E., Los Angeles, CA 90095. Tel.: 310-794-5763; Fax: 310-794-5766; E-mail: arispe@mbi.ucla.edu.
↵2 F. Vázquez, G. Hastings, M.-A. Ortega, T. F. Lane, S. Oikemus, M. Lombardo, and M. L. Iruela-Arispe, unpublished observations.
Abbreviations:

TSP1

thrombospondin-1

pNPI

procollagen 1 N-proteinase

GPDH

glyceraldehyde-3-phosphate dehydrogenase

CAM

chorioallantoic membrane

FGF-2

fibroblast growth factor-2

VEGF

vascular endothelial growth factor

HDEC

human dermal endothelial cell

kb

kilobase pair(s)

DMEM

Dulbecco’s modified Eagle’s medium

ADAM

a disintegrin andmetalloprotease

PAGE

polyacrylamide gel electrophoresis

EBM

endothelial cell basal media
- Received April 22, 1999.
- Revision received May 24, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.