V D Bhoyroo

Molecular cloning and expression of rat liver endo-alpha-mannosidase, an N-linked oligosaccharide processing enzyme.

A clone containing the open reading frame of endo-α-d-mannosidase, an enzyme involved in earlyN-linked oligosaccharide processing, has been isolated from a rat liver λgt11 cDNA library. This was accomplished by a strategy that involved purification of the endomannosidase from rat liver Golgi by ligand affinity chromatography (Hiraizumi, S., Spohr, U., and Spiro, R. G. (1994) J. Biol. Chem. 269, 4697–4700) and preparative electrophoresis, followed by sequence determinations of tryptic peptides. Using degenerate primers based on these sequences, the polymerase chain reaction with rat liver cDNA as a template yielded a 470-base pair product suitable for library screening as well as Northern blot hybridization. EcoRI digestion of the purified λ DNA released a 5.4-kilobase fragment that was amplified in Bluescript II SK(−) vector. Sequence analysis indicated that the deduced open reading frame of the endomannosidase extended from nucleotides 89 to 1441, encoding a protein of 451 amino acids and corresponding to a molecular mass of 52 kDa. Data base searches revealed no homology with any other known protein. When a vector coding for this protein fused to an NH2-terminal peptide containing a polyhistidine region was introduced intoEscherichia coli, high levels of the enzyme were expressed upon induction with isopropyl-β-d-thiogalactoside. Purification of the endomannosidase to electrophoretic homogeneity fromE. coli lysates was accomplished by Ni2+-chelate and Glcα1→3Man-O-(CH2)8CONH-Affi-Gel ligand chromatographies. Polyclonal antibodies raised against this protein reacted with Golgi endomannosidase. By both immunoblotting and silver staining, the purified E. coli-expressed enzyme was approximately 8 kDa smaller than anticipated from the open reading frame; timed induction studies indicated that this was due to scission of the enzyme’s COOH-terminal end by host cell proteases. All rat tissues examined demonstrated mRNA levels (4.9-kilobase message) for the endomannosidase that correlated well with their enzyme activity.