Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form.
A cDNA was cloned coding for human placental 5'-nucleotidase. The 3547-bp cDNA contains an open reading frame that encodes a 574-residue polypeptide with calculated size of 63 375 Da. The NH2-terminal 26 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5'-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 510-523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the mature 5'-nucleotidase lacks the predicted COOH-terminal peptide extension (524-548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5'-nucleotidase.
Misumi
Y
Y
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Ogata
S
S
Ohkubo
K
K
Hirose
S
S
Ikehara
Y
Y
D015720:5'-Nucleotidase / Q000032:analysis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D006017:Glycolipids / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments; D010716:Phosphatidylinositols / Q000032:analysis; D010920:Placenta / Q000201:enzymology*; D015183:Restriction Mapping
D006017:Glycolipids; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D004247:DNA; D015720:5'-Nucleotidase; D003488:Cyanogen Bromide