Structural and functional analysis of human germ cell alkaline phosphatase by site-specific mutagenesis.
Human germ cell alkaline phosphatase (GCAP), which shares 98% amino acid sequence identity with the placental AP (PLAP), is expressed by malignant trophoblasts. Protein sequence analysis suggests that the Ser residue at position 92 is the putative active site of GCAP which contains two recognition sequences (Asn122-Thr-Thr124 and Asn249-Arg-Thr251) for asparagine-linked glycosylation. To examine the roles of the Ser residue and glycan moieties on GCAP activity and processing, we altered the GCAP cDNA by site-directed mutagenesis and expressed the GCAP mutants in COS-1 cells. Substitution of Ser-92 with either a Thr (S92T) or an Ala (S92A) residue yielded a GCAP devoid of catalytic activity, suggesting that the Ser codon 92 is the active site of GCAP. Six GCAP mutants that lack one or both glycosylation sites were constructed by substituting either Asn-122 or Asn-249 with an Asp residue or either Thr-124 or Thr-251 with an Ala residue. The mature GCAP migrated as a 65-kDa product, but GCAP mutants lacking one or both glycosylation sites migrated as 62- or 58-kDa polypeptides, respectively, indicating that both sites were glycosylated. All six glycosylated mutants were active enzymatically and, in addition, were equally sensitive to heat, L-leucine, and EDTA inhibition as the parental enzyme. GCAP as well as its two active-site and six glycosylation mutants could be released from the plasma membrane of transfected COS-1 cells by the proteinase bromelain.(ABSTRACT TRUNCATED AT 250 WORDS)
Watanabe
T
T
Human Genetics Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Wada
N
N
Chou
J Y
JY
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D005854:Germ Cells / Q000737:chemistry / Q000201:enzymology; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007668:Kidney; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D008565:Membrane Proteins; C055791:germ-cell AP isoenzyme; D012694:Serine; D000469:Alkaline Phosphatase