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-alpha-D-mannosidase, an enzyme involved in early N-linked oligosaccharide processing, has been isolated from a rat liver lambdagt11 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 lambda 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 into Escherichia coli, high levels of the enzyme were expressed upon induction with isopropyl-beta-D-thiogalactoside. Purification of the endomannosidase to electrophoretic homogeneity from E. coli lysates was accomplished by Ni2+-chelate and Glcalpha1-->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.
Spiro
M J
MJ
Department of Biological Chemistry, Harvard Medical School and the Joslin Diabetes Center, Boston, Massachusetts 02215, USA.
Bhoyroo
V D
VD
Spiro
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D006056:Golgi Apparatus / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D016366:Open Reading Frames; D010449:Peptide Mapping; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D004274:DNA, Recombinant; D009844:Oligosaccharides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008361:Mannosidases; C051635:endo-alpha-D-mannosidase; D014357:Trypsin