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Mouse ADAM33

Two Splice Variants Differ in Protein Maturation and Localization

Departments of Allergy and of Statistics, Schering-Plough Research Institute, Kenilworth, New Jersey; and Genome Therapeutics Corporation, Waltham, Massachusetts

Address correspondence to: Shelby P. Umland, Ph.D., Schering-Plough Research Institute, 2015 Galloping Hill Road, K15-1-1700, Kenilworth, NJ 07033. E-mail: shelby.umland{at}spcorp.com

We compared the tissue mRNA prevalence and protein maturation of two splice variants of mouse ADAM33, a metalloprotease implicated in airway hyperresponsiveness. These variant cDNAs, designated 914 () and 906 (ß), encode membrane-bound forms that differ primarily in 26 residues (exon 17) between the cysteine-rich and epidermal growth factor–like domains. Proteins of 120 and 103 kD, detectable by anti-ADAM33 antibodies, were expressed in 914-transfected HEK293 cells. The time-dependent appearance of the 100-kD form and its inhibition by a peptidyl chloromethylketone, or the calcium ionophore, A23187, indicated that this was mature ADAM33, which was processed by a furin-like convertase. One form, 110 kD, was detected in 906-transfected cell lysates. Trypsin and biotinylation treatment of transfected cells demonstrated that all of the mature 100-kD, a minority of the 120-kD pro-form, and none of the 906-expressed 110-kD form localized to the cell surface. The mature form was resistant to endoglycosidase H_f. The 110-kD form was endoglycosidase H_f-sensitive, indicating retention proximal to the trans-Golgi, consistent with a lack of maturation. Quantitation of transcripts demonstrated that those containing exon 17 predominate, whereas those lacking exon 17 are negligible in the mouse lung, although detectable at low levels in mouse testis, heart, and brain. Thus, potential dominant-negative effects exerted by the nonprocessed 906-encoded ß splice variant are unlikely to occur in mouse lung.

Abbreviations: antibody, Ab • a disintegrin and metalloprotease, ADAM • dec-Arg-Val-Lys-Arg-chloromethyl ketone, decRVKR • epidermal growth factor, EGF • endoglycosidase H_f–resistant, Endo H^r • endoglycosidase H_f–sensitive, Endo H^s • endoplasmic reticulum, ER • human embryonic kidney cells, HEK • histidine, His • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • peptide:N-glycosidase F-resistant, PNGase F^r • peptide:N-glycosidase–sensitive, PNGase F^s • treatment, Rx • streptavidin, SA • Tris-buffered saline, TBS • TBS with 0.1% Tween 20, TBS-T