2 NATURALLY-OCCURRING MOUSE ALPHA-1,2-MANNOSIDASE IB CDNA CLONES DIFFER IN 3 POINT MUTATIONS - MUTATION OF PHE(592) TO SER(592) 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 cy-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 (Thr(411), Leu(468), and Ser(592) in clone 4 to Met(411), Phe(468), and Phe(592) 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 [H-3]mannose-labeled Man(9)GlcNAc 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 Phe(592) to Ser(592), as found in clone 4, is sufficient to abolish alpha-1,2-mannosidase activity, whereas mutation of Met(411) to Thr(411) or of Phe(468) to Leu(468) affects secretion with relatively little effect on enzyme activity. Phe(592) is part of a highly conserved region that seems important for enzyme activity of class 1 alpha-1,2-mannosidases.