accuracy of theoretical mass value
accuracy of theoretical mass value. glycotopes on LAMP-1 was reduced in FUT1 knockdown cells as compared with those of the mock or control cells. Similar to the tandem mass analysis of H2 and LeX structural isomer, the B-fragment ions (834.4) in MS/MS spectrum, which could only be found in bi-antennary glycans with three fucose residues, was sijmilarly selected at the retention time 24.1?min for MS3 analysis of LeY glycotopes. The identification of LeY was mainly based on diagnostic fragment ions at 415, 433, 646 and cross-ring fragment ions at 503 (3,5A) in the MS3 spectrum. Similar 4-Aminobenzoic acid to H2 glycotopes, EIC of 884.8 for the representative glycans of LAMP-1 showed a decrease in the intensity of LeY glycotopes upon FUT1 knockdown. This is consistent with our result in Figure 1a that this expression of LeY on LAMP-1 was reduced upon FUT1 knockdown. Taken together, these MS results further verified that FUT1 is responsible for the terminal fucosylation of H2 and LeY HOXA11 found on both LAMP-1 and 2. Supplementary Table S1 summarizes the results of fucosylation changes in LAMP-1 or LAMP-2 upon FUT1 knockdown detected by various analytical methods. Open in a separate window Physique 2 Characterization of 1141.6, [M+2Na]2+), two (826.7, [M+3Na]3+) or three (884.8, [M+3Na]3+) fucose residues of purified LAMP-1 from mock, control or FUT1 knockdown cells. The EICs were reconstructed by ion intensities within 20?p.p.m. accuracy of theoretical mass value. The major fragment ions of H type 2 (H2), Lewis X (LeX) and LeY glycotopes are schematically illustrated on the right panel. Notably, those bi-antennary 2709, 2883 and 3057 for tri-antennary and 3158, 3332 and 3506 for tetra-antennary N-glycans, respectively. The relative ratio of each glycoform is given in percentage of total sum of peak intensities of tri- and tetra-antennary glycans in the MS spectra. The colored symbol and nomenclature for glycan structure are based on the designation of Consortium for Functional Glycomics as described in Physique 2a. Peaks labeled with asterisks represent polyhexose ladder contaminations that were negligible for overall analysis Downregulation of FUT1 leads to accumulation of LAMP-1/2(+) vesicles at perinuclear area Upon silencing of FUT1 in MCF-7 and T47D breast malignancy cells, we observed a striking change in the subcellular distribution patterns of LAMP-1 and 2 by immunofluorescence staining. As shown in Physique 4a, LAMP-1 staining in the control cells appeared as vesicle-like structures and distributed randomly in the cytoplasm. In contrast, LAMP-1(+) vesicles in FUT1 knockdown cells mostly accumulated in the perinuclear region. Quantitative analysis showed that this proportion of cells with predominantly perinuclear LAMP-1(+) vesicles increased from 17.160.1% and 52.275.3% in control MCF-7 and T47D cells, respectively, to 61.393% and 87.934.4% in FUT1 silenced MCF-7 and T47D cells (that LAMP-1 is identified as a carrier for LeY antigens, our 4-Aminobenzoic acid study has additionally demonstrated the presence of H2 and LeY antigens on LAMP-1 is mediated by FUT1 but not FUT2. Similarly, we have also identified the LAMP-1 family member, LAMP-2, as 4-Aminobenzoic acid a novel substrate of FUT1 with LeY moiety attached. Topographically, we have discovered a striking change in the subcellular localization 4-Aminobenzoic acid of LAMP-1 and 2 upon FUT1 knockdown in which LAMP-1 and 2 were preferentially accumulated at perinuclear region rather than being at the peripheral region, as seen in the control cells. On the other hand, we have found that knockdown of FUT1 results in an increased rate of autophagosome formation and degradation, which is accompanied by a decrease in mTORC1 (a 4-Aminobenzoic acid known suppressor of autophagy) activity and an increase in autophagosomeClysosome fusion. As lysosomal positioning has been reported to coordinate mTOR activity and autophagy, the improvement of autophagic flux in FUT1 knockdown cells is apparently the consequence of reduced mTOR signaling and improved autolysosome development. Although LeY transported by surface.