cDNA encoding a functional feline liver/bone/kidney-type alkaline phosphatase

Arch Biochem Biophys. 1995 Sep 10;322(1):240-9. doi: 10.1006/abbi.1995.1458.

Abstract

Feline alkaline phosphatase (FeALP) was copurified with the putative 70-kDa feline leukemia virus subgroup-A (FeLV-A) receptor protein from feline T-lymphocyte cells (FeT) by two-dimensional gel electrophoresis. The sequence of the N-terminal 17 amino acids and five other internal tryptic peptides revealed that it is homologous to the liver/bone/kidney (L/B/K)-type alkaline phosphatase of other mammalian species. Corresponding oligonucleotides were synthesized and used for amplification of a 1.2-kb segment of the FeALP gene by polymerase chain reaction, using phage DNA from a FeT cell cDNA library as template. The 1.2-kb FeALP gene fragment generated was then used as a probe to isolate a 2127-bp L/B/K-type FeALP cDNA clone from the same library harboring a large, intact open reading frame. This cDNA possessed an open reading frame encoding a 524-amino-acid protein including a putative signal peptide of 17 amino acids followed by 14-amino-acid residues identical to the N-terminal sequence determined from the purified protein. Sequences closely related to five tryptic peptides from the purified protein were also contained within the cDNA-encoded protein. Homology with the human, bovine, rat and mouse L/B/K-type ALP was found to be 88-90% at both the nucleotide and the amino acid levels. The cDNA was transferred into a eukaryotic expression vector and expressed following transfection into murine and mink lung fibroblast cell lines. High levels of enzymatically active ALP were detected, along with a 70-kDa protein reactive in immunoblot assay using a polyclonal antibody against the original crude FeALP preparation. FeALP was specifically released from intact cells by treatment with phosphoinositol-specific phospholipase-C. By Northern blot analysis, only one species of mRNA was detected using a 32P-labeled cDNA probe. These results indicate that the 2127-bp cDNA encodes a functional feline L/B/K-type ALP expressed on cell surfaces via phosphatidylinositol-glycan linkage. Despite electrophoretic comigration in two dimensions and following deglycosylation in a third dimension, FeALP failed to function as an FeLV receptor since its expression failed to provide for attachment or entry of virus into cells.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkaline Phosphatase / genetics*
  • Alkaline Phosphatase / isolation & purification
  • Alkaline Phosphatase / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Bone and Bones / enzymology
  • Cats
  • Cell Line
  • Cell Membrane / enzymology
  • Cloning, Molecular
  • DNA Primers / genetics
  • DNA, Complementary / genetics*
  • Humans
  • Kidney / enzymology
  • Leukemia Virus, Feline / metabolism
  • Liver / enzymology
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Receptors, Virus / metabolism
  • Sequence Homology, Amino Acid
  • Species Specificity
  • Tissue Distribution

Substances

  • DNA Primers
  • DNA, Complementary
  • Receptors, Virus
  • Alkaline Phosphatase

Associated data

  • GENBANK/U31569