Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

The murine Man II coding sequence predicts a polypeptide of 1,150 amino acids (M, 131,000) with a single type II The Joumal of Cell Biology, Volume 115, 1991 Figure 3 In vitro transcription/ translation of Man II clone MII-8 and comparisonwithbiosynthetically labeled Man II. Man II clone MII-8 was transcribed in vitro using T7 polymerase as described in Materials and Methods . In vitro translation was performed with or without the addition of microsomal membranes (co-or posttranslational addition of membranes) as indicated at the bottom of the figure . Aliquots of the translation were thendigested or mock digested with trypsin in the presence or absence of Triton X-100 as indicated . Samples were resolved by SDS-PAGE and subjected to autoradiography. Lanes A and B represent cell extracts from biosynthetically labeled 3T3 cells which were immunoprecipitated with the anti-Man II antibody and either digested (B) or mock digested (A) with N-glycanase . Samples were resolvedon the same gel as the in vitro translation samples . Lanes A and B were exposed to x-ray film for 3 d while the in vitro translation samples were exposed for 12 h . Identical results for the in vi immunoprecipitated with anti-Man II antibody following the synthesis and pro-kD) are indicated at the left of the figure . transmembrane domain from amino acid residue 6-26. Several lines of evidence suggest that the coding region shown in Fig . 2 contains the correct initiation site for the Man II open reading frame. The Met codon in position 1 is the first ATG in the sequence and conforms to the consensus eucaryotic translation sequence with a purine at position -3, the most critical residue in the initiation sequence (19) . The 5' untranslated region is G/C rich (68%), a characteristic common to several of the Golgi glycosyltransferases (46, 53) and also a common feature among housekeeping genes (9) . Finally, the NHZ-terminal peptide sequence of the purified intact rat liver enzyme starts at residue 6 of the predicted open reading frame (30) . The purified enzyme is indistinguishable in size from the biosynthetically labeled 3T3 enzyme (27) and the enzyme detected by Western blots from freshly prepared Golgi membrane extracts (27), but the cleavage of five residues would be too small a change to detect on SDS gels during purification . Although the cleavage of the five residue cytoplasmic tail most likely reflects in vitro proteolysis during purification, it could also possibly represent the product of cleavage in vivo .