Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

Abstract

The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 ('a disintegrin and metalloprotease 28'), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal.