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 a calculated size of 63375 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is concluded that the mature 5′‐nucleotidase lacks the predicted COOH‐terminal peptide extension (524–548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5′‐nucleotidase. Copyright © 1990, Wiley Blackwell. All rights reserved