Human tissue non-specific alkaline phosphatases: sugar-moiety-induced enzymic and antigenic modulations and genetic aspects.
To investigate the possible role(s) of glycans in human tissue non-specific alkaline phosphatase (TNAP) activity, the iso-enzymes were purified and treated with various exo- and endo-glycosidases. Catalytic activity, oligomerization, conformation and immunoreactivity of the modified TNAPs were evaluated. All TNAPs proved to be N-glycosylated, and only the liver isoform (LAP) is not O-glycosylated. Usually, the kidney (KAP) and bone (BAP) isoenzymes are similar and cannot be clearly discriminated. Differences between the immunoreactivity of KAP/BAP and LAP with a BAP antibody were mainly attributed to the N-glycosylated moieties of the TNAPs. In addition, elimination of O-glycosylations moderately affects the TNAP reactivity. Interestingly, N-glycosylation is absolutely essential for TNAP activity, but not for that of the placental or intestinal enzymes. According to the deduced amino acid sequence of TNAP cDNA, Asn-213 is a possible N-glycosylation site, and our present findings suggest that this sugar chain plays a key role in enzyme regulation. With regard to the oligomeric state of alkaline phosphatase (AP) isoforms, the dimer/tetramer equilibrium is dependent on the deglycosylation of glycosyl-phosphatidylinositol(GPI)-free APs, but not GPI-linked APs. This equilibrium does not affect the AP conformation as observed with CD. With regard to TNAPs, no data were available on the gene expression or nature of the 5'-non-translated leader exon of human KAP, as opposed to BAP and LAP genes. cDNA sequencing revealed that cortex/medulla KAP is genetically related to BAP, and medulla KAP to LAP.
Nosjean
O
O
Laboratoire de Physico-Chimie Biologique, URA-CNRS 1535, Université Claude Bernard-Lyon, Villeurbanne, France.
Koyama
I
I
Goseki
M
M
Roux
B
B
Komoda
T
T
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D001483:Base Sequence; D001842:Bone and Bones / Q000201:enzymology; D018076:DNA, Complementary / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010513:Periodontal Ligament / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis; D013379:Substrate Specificity
D018076:DNA, Complementary; D012333:RNA, Messenger; D000469:Alkaline Phosphatase