FIGURE 4.

All four putative sites in ADAM8 are N-glycosylated. A, schematic representation of ADAM8-Myc/His protein with the different domains and putative N-glycosylation sites indicated. N-Glycosylation sites were individually mutated from Asn to Gln in the pCDNA 3.1/Myc-His (−)Ver B vector, which expresses proteins with a C-terminal Myc/His tag. Pro: prodomain; MP: metalloproteinase domain: DI: disintegrin domain; CRD: cysteine-rich domain; ELD: EGF-like domain; TM: transmembrane domain; CTD: cytoplasmic domain. B, representation of the predicted three-dimensional model of ADAM8 (195–647 residues including MP, DI, CRD, and ELD). N-Glycosylation residues 436 and 612 are highlighted in blue. Figures produced using PyMol. C and D, HEK-293 (C) or MDA-MB-231 (D) cells were transiently transfected with empty vector (EV) DNA, or with vectors expressing Myc-His-tagged WT ADAM8 or N67Q, N91Q, N436Q, or N612Q N-glycosylation mutants. Cells were lysed after 24 h, and samples subjected to immunoblot analysis of ADAM8 expression, as above, using a c-Myc antibody to detect ectopically expressed proteins. The white dashed line indicates the migratory position of the proform expressed by WT ADAM8.