title: N-linked glycosylation of dipeptidyl peptidase IV (CD26): effects on enzyme activity, homodimer formation, and adenosine deaminase binding.
abstract: The type II transmembrane serine protease dipeptidyl peptidase IV (DPPIV), also known as CD26 or adenosine deaminase binding protein, is a major regulator of various physiological processes, including immune, inflammatory, nervous, and endocrine functions. It has been generally accepted that glycosylation of DPPIV and of other transmembrane dipeptidyl peptidases is a prerequisite for enzyme activity and correct protein folding. Crystallographic studies on DPPIV reveal clear N-linked glycosylation of nine Asn residues in DPPIV. However, the importance of each glycosylation site on physiologically relevant reactions such as dipeptide cleavage, dimer formation, and adenosine deaminase (ADA) binding remains obscure. Individual Asn-->Ala point mutants were introduced at the nine glycosylation sites in the extracellular domain of DPPIV (residues 39-766). Crystallographic and biochemical data demonstrate that N-linked glycosylation of DPPIV does not contribute significantly to its peptidase activity. The kinetic parameters of dipeptidyl peptidase cleavage of wild-type DPPIV and the N-glycosylation site mutants were determined by using Ala-Pro-AFC and Gly-Pro-pNA as substrates and varied by <50%. DPPIV is active as a homodimer. Size-exclusion chromatographic analysis showed that the glycosylation site mutants do not affect dimerization. ADA binds to the highly glycosylated beta-propeller domain of DPPIV, but the impact of glycosylation on binding had not previously been determined. Our studies indicate that glycosylation of DPPIV is not required for ADA binding. Taken together, these data indicate that in contrast to the generally accepted view, glycosylation of DPPIV is not a prerequisite for catalysis, dimerization, or ADA binding.
authors.affiliation: Syrrx Inc., San Diego, California 92121, USA. kathleen.aertgeerts@syrrx.com
authors.forename: Kathleen
authors.initials: K
authors.lastname: Aertgeerts
authors.forename: Sheng
authors.initials: S
authors.lastname: Ye
authors.forename: Lihong
authors.initials: L
authors.lastname: Shi
authors.forename: Sridhar G
authors.initials: SG
authors.lastname: Prasad
authors.forename: Darbi
authors.initials: D
authors.lastname: Witmer
authors.forename: Ellen
authors.initials: E
authors.lastname: Chi
authors.forename: Bi-Ching
authors.initials: BC
authors.lastname: Sang
authors.forename: Robert A
authors.initials: RA
authors.lastname: Wijnands
authors.forename: David R
authors.initials: DR
authors.lastname: Webb
authors.forename: Ronald V
authors.initials: RV
authors.lastname: Swanson
mesh_terms: D000243:Adenosine Deaminase / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D019281:Dimerization; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004220:Disulfides; D018819:Dipeptidyl Peptidase 4; D000243:Adenosine Deaminase; D000409:Alanine