Two naturally occurring mouse alpha-1,2-mannosidase IB cDNA clones differ in three point mutations. Mutation of Phe592 to Ser592 is sufficient to abolish enzyme activity.
In mammalian cells, alpha-1,2-mannosidases play an essential role in the early steps of N-linked oligosaccharide maturation. We previously reported (Herscovics, A., Schneikert, J., Athanassiadis, A., and Moremen, K. W. (1994) J. Biol. Chem. 269, 9864-9871) the isolation of mouse alpha-mannosidase IB cDNA clones from a Balb/c 3T3 cDNA library. Clone 4 encodes a type II membrane protein of 641 amino acids with a cytoplasmic tail of 35 amino acids, followed by a transmembrane domain and a large C-terminal catalytic domain, whereas clone 16 encodes only the last 471 amino acids. Their overlapping sequences (from amino acid 152) are identical, except for three point mutations that result in three amino acid differences in the catalytic domain of the enzyme (Thr411, Leu468, and Ser592 in clone 4 to Met411, Phe468, and Phe592 in clone 16, respectively). Both sequences could be amplified by polymerase chain reaction using templates of cDNAs derived from colon and brain of CD1 mice and from L cells derived from the C3H/An mouse, indicating that both are natural isoforms found in two inbred and one outbred mouse strains. When expressed in COS7 cells as a secreted protein A fusion protein, the catalytic domain of clone 16 displays alpha-1,2-mannosidase activity using [3H]mannose-labeled Man9GlcNAc as substrate, but the corresponding region of clone 4 is poorly secreted under identical conditions. The contribution of each point mutation to this differential secretion and enzyme activity of the two fusion proteins was assessed by testing the six recombinants corresponding to all the possible sequence permutations. Mutation of Phe592 to Ser592, as found in clone 4, is sufficient to abolish alpha-1,2-mannosidase activity, whereas mutation of Met411 to Thr411 or of Phe468 to Leu468 affects secretion with relatively little effect on enzyme activity. Phe592 is part of a highly conserved region that seems important for enzyme activity of class 1 alpha-1,2-mannosidases.
Schneikert
J
J
McGill Cancer Centre, McGill University, Montréal, Québec, Canada.
Herscovics
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D012694:Serine; D010649:Phenylalanine; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase