Human ADAM33: protein maturation and localization

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Abstract

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene [18]. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an α2-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.

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Materials and methods

Reagents. CHO-K1, COS-7, and MRC-5 cell lines were obtained from the American Type Culture Collection (Rockville, MD), HEK-293-D22 cells were obtained from Canji (San Diego, CA), and human bronchial smooth muscle cells were obtained from Clonetics Cell Discovery Systems (BioWhittaker, Walkersville, MD). Human bronchus was obtained from Tissue Transformation Technologies (Edison, NJ) with the appropriate legal consent of the donor and/or the donor’s next of kin. KB8301 was from PharMingen (BD

Catalytic activity of the metalloprotease domain

Because ADAM33 substrates are currently unknown, it was not possible to test full-length, membrane-bound protein for protease activity in cell-based cleavage assays. However, it was possible to demonstrate activity using a soluble form of the enzyme in combination with α2M, a substrate known to be cleaved by most proteases [19], [20].

A cell line was developed that expressed a soluble form of the ADAM33 catalytic domain with a carboxyl-terminus at aspartic acid 432 followed by a six-histidine

Discussion

ADAM33 contains five potential sites for asparagine-linked (N-linked) oligosaccharide modification; two sites within the pro-domain, two sites within the catalytic domain, and one site within the disintegrin domain [17 and computer analysis not shown]. Analysis of full-length ADAM33 for oligosaccharide content indicated that the immature protein consisted of predominantly high-mannose or hybrid oligosaccharides. As the protein matured, some but perhaps not all of the oligosaccharides were

Acknowledgements

The authors thank Dr. C.W. McNemar (Schering-Plough Research Institute, SPRI) for determination of N-terminal amino acids of recombinantly expressed ADAM33 and C. Rizzo (SPRI) for procurement of the human bronchus tissue. We thank Dr. L. Xiao and Dr. V. Madison (SPRI) for help in designing peptides for antibody generation and Dr. R. Ralston (Canji) for the gift of HEK-293-D22 cells. We thank Dr. C. Carlson (Covance Research Products Inc.), Dr. S.-E. Yen (Zymed Laboratories Inc.), and G. Ruiz

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