Roles of cysteines in rat dipeptidyl peptidase IV/CD26 in processing and proteolytic activity.
The multifunctional type II transmembrane glycoprotein, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), is expressed by almost all mammalian cells and is identical to the adenosine deaminase binding protein CD26 on lymphocytes. The extracellular part of rat DPPIV can be divided into three domains the middle part of which harbors 10 of the 12 highly conserved cysteine residues. The cysteine-rich domain is responsible for DPPIV-binding to collagen I and to extracellular ADA. The participation of distinct cysteines in disulfide bridges is not yet known. Titration experiments have shown the presence of six free cysteines and three disulfide bridges in native rat DPPIV. To investigate the role of distinct cysteines in the structure-function relationships of rat DPPIV we constructed 12 different cysteine point mutations (C299, C326, C383, C455, C650 mutated to G; C337, C395, C445, C448, C473, C552, C763 mutated to S). Intracellular translocation to the cell surface of stable transfected Chinese hamster ovary cells was examined with antibodies against different epitopes of DPPIV. Surface expression of mutants C326G, C445S and C448S is inhibited totally; mutants C337S, C455G, C473S and C552S show weak expression only. In parallel, the half-life of these mutants is reduced to < 10% compared with wild-type enzyme. We were able to show that the specific peptidase activity of the mutant protein depends on cell-surface expression, dimerization and the existence of a 150-kDa form demonstrable by nondenaturing SDS/PAGE. We conclude that cysteine residues 326, 337, 445, 448, 455, 473 and 552 in rat DPPIV are essential for the correct folding and intracellular trafficking of this glycoprotein, and therefore for its normal biological properties.
Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Germany.
J
J
Dobers
S
S
Grams
W
W
Reutter
H
H
Fan
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis
D011994:Recombinant Proteins; D018819:Dipeptidyl Peptidase 4; D003545:Cysteine