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Human ADAM33 Messenger RNA Expression Profile and Post-Transcriptional Regulation

Departments of Allergy and Human Genomics, Schering-Plough Research Institute, Kenilworth, New Jersey; and Canji Inc., San Diego, California

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 examined transcript expression and post-transcriptional regulation of human ADAM33, a recently identified asthma gene. A detailed messenger RNA (mRNA) expression profile was obtained using Northern, reverse transcription polymerase chain reaction, and in situ hybridization analyses. ADAM33 mRNA was expressed significantly in smooth muscle–containing organs, minimally in immune organs and hematopoietic cells, and highly in repairing duodenal granulation tissue. Expression was seen in asthmatic subepithelial fibroblasts and smooth muscle but not in respiratory epithelium. In all tissues, transcripts of 5 kb predominated over those of 3.5 kb by 2- to 5-fold. The effect of the 3' untranslated region (UTR) on ADAM33 protein expression and maturation was examined. The presence of the 3'UTR in untagged full-length constructs promoted prodomain removal, detected as mature 100 kD protein by ADAM33-reactive antibodies; in its absence, maturation was 2- to 3-fold less in HEK293 cells. His-tagged and untagged constructs lacking the 3'UTR demonstrated that lack of maturation was not a result of tag-mediated effects. Minimal maturation of ADAM33 occurred in primary lung and MRC5 fibroblasts following adenoviral-mediated expression of ADAM33 lacking the 3'UTR. In contrast, prodomain removal was observed with plasmids and adenovirus encoding only the pro- and catalytic domains. Thus, the 3'UTR of ADAM33 and domains downstream of the catalytic domain regulate potential ADAM33 activity. Mechanisms of regulation of ADAM33, distinct from closely related ADAMs, thus include mRNA localization and processing and protein maturation.

Abbreviations: antibody, Ab • a distintegrin and metalloprotease, ADAM • bronchial smooth muscle cells, BSMC • complimentary DNA, cDNA • Chinese hamster kidney cells, CHO-K1 • cycle threshold, Ct • epidermal growth factor, EGF • human embryonic kidney cells, HEK • a tag of six histidine residues, his • messenger RNA, mRNA • normal human lung fibroblasts, NHLF • in situ hybridization, ISH • polymerase chain reaction, PCR • recombinant adenovirus, rAd • reverse transcription PCR, RT-PCR • Tris-buffered saline, TBS • TBS with 0.1% Tween 20, TBS-T • tumor necrosis factor, TNF • untranslated region, UTR