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

Immunocytochernistry at the EM level using antibodies to purified rat Man II has demonstrated the Golgi localization of the enzyme in rat tissues (44) . The antibodies have also been used recently as a marker for Golgi membrane components in cell trafficking studies (8, 22; and C. Zuber, A. Nakano, K. W. Moremen and J. Roth, manuscript submitted for publication) . Preliminary studies have shown that the antibody raised to the rat enzyme does not cross react with the COS cell enzyme (data not shown) allowing a minimal immunofluorescence background in the host cells. Transfection of COS cells with the pXM construct containing the Man II insert in the sense orientation (MII-pXM) resulted in an anti-Man II immunofluorescence pattern within 24 h ofthe initiation oftransfection. The immunofluorescence was largely restricted to a crescent shaped reticular pattern adjacent to the nucleus, but occasionally this reticular structure would extend well into the cytoplasm (Fig. 7, A and B ). This pattern of immunofluorescence has been previously shown to be characteristic ofthe Golgi complex (22, 31, 44) . Mock transfections, transfections with vector alone, or with the Man II insert in the antisense orientation resulted in no detectable immunofluorescence signal (data not shown) . Similarly, immunofluorescence offixed but not permeabilized cells transfected with MII-pXM resulted in no detectable fluorescence signal . The transfection efficiency ofthe MII-pXM construct, as measured by thepercentage of fluorescence-positive cells, was variable, but ranged from 5-25 R . By 36 h posttransfection the intensity of the peri-nuclear immunofluorescence increased substantially with many cells generating an additional diffuse particulate pattern of fluorescence in the cytoplasm consistent with ER staining (8, 22, 31) (Fig. 7 , C-E) . By 48 h postuansfection immunofluorescence staining adjacent to the nucleus remained intense but the particulate cyto- The human chromosome compliment of each hybrid was compared with the ability to generate the 178-bp human Man II amplification fragment following PCR. Concordant segregationof the amplification fragment was observed only for chromosome 5. All other chromosomes were excluded as the site for the Man II gene by discordancies in at least 15 hybrids . Hybrid clones containing the indicated chromosome in at least 5% of the cells were considered positive for the chromosome in the discordancy analysis . plasmic staining greatly increased . With the high level of Man II synthesis late in the transient expression, detection of early biosynthetic intermediates in the ER or an accumulation of aggregated enzyme in the ER might be expected.