Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase.
Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.
Proudfoot
A E
AE
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Turcatti
G
G
Wells
T N
TN
Payton
M A
MA
Smith
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism*; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D005800:Genes, Fungal; D006801:Humans; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology*; D010957:Plasmids; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008359:Mannose-6-Phosphate Isomerase