Inhibition of Bone Morphogenetic Protein 1 by Native and Altered Forms of α2-Macroglobulin

Yue Zhang,Gaoxiang Ge,Daniel S Greenspan

doi:10.1074/jbc.m601362200

Yue Zhang, Gaoxiang Ge + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m601362200

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Abstract

The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein alpha(2)-macroglobulin is unable to inhibit the astacin-like proteinases meprin alpha and meprin beta, we herein demonstrate alpha(2)-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the alpha(2)-macroglobulin "bait" region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I-III. alpha(2)-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified alpha(2)-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced approximately 24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of alpha(2)-macroglobulin in the treatment of fibrotic conditions.

Highlights

This class of proteinases activate a subset of the transforming growth factor-␤ superfamily of proteins in a broad range of species, through cleavage of extracellular protein antagonists [2]
A genetically altered version of ␣2M, in which the bait region has been replaced by the bone morphogenetic protein 1 (BMP1) cleavage site of the small leucine-rich proteoglycan precursor probiglycan is shown to have a greatly enhanced ability to inhibit BMP1, and is able to inhibit procollagen I processing by cells
BMP1-like proteinases are important regulators of ECM deposition in vertebrates. They are of central importance to the formation of collagen fibrils because 1) they

Summary

EXPERIMENTAL PROCEDURES

Cleavage of ␣2M by BMP1—200 nM FLAG-tagged recombinant BMP1, prepared, and purified as previously described [11], was incubated overnight at 37 °C with 200 nM human ␣2M (Sigma) in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10 mM CaCl2. The appearance of pN␣1(I) chains in the wild type ␣2Mtreated sample and procollagen in the b-␣2M-treated sample indicate that both forms of ␣2M are able to inhibit N-propeptide cleavage in cell culture by the proteinase ADAMTS-2. Time course experiments conducted with a fixed ment of MC-3T3-E1 cells with plasma ␣2M (data not shown), amount of 35S-radiolabeled BMP1 (40 nM) incubated with increas- yielded effects on procollagen processing similar to those ing amounts of each form of ␣2M, as in Enghild et al [14], provided Ki values of 36.4, 39.5, and 25.2 nM for b-␣2M, and wild type recombinant and plasma ␣2M, respectively (Fig. 7). Obtained from treatment of cells with recombinant wild type ␣2M

DISCUSSION

Findings

Relative rate constant effectiveness