The poly-N-acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex.
The poly-N-acetyllactosamines on neutrophils and monocytes have been shown to serve as ligands for various selectins present on endothelial cells and platelets. We have previously shown that only a limited number of glycoproteins contain poly-N-acetyllactosamine and found that lysosomal membrane glycoproteins (lamps) are the major glycoproteins carrying poly-N-acetyllactosamine. In order to understand the reason why only certain glycoproteins can be modified by poly-N-acetyllactosamine, we have utilized 21 degrees C incubation conditions, which were previously shown to cause the accumulation of glycoproteins at the trans-Golgi. HL-60 cells were labeled with [3H]galactose at 21 or 37 degrees C for 6 or 24 h, and lamp-1 and lamp-2 were immunoprecipitated. Upon examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, each lamp from HL-60 cells incubated at 21 degrees C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37 degrees C. The number of N-glycans containing poly-N-acetyllactosamine, estimated by their binding to tomato lectin column, increased approximately 30-50% after incubation at 21 degrees C than incubation at 37 degrees C. The analysis of oligosaccharides released by endo-beta-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100% after incubation at 21 degrees C, and methylation analysis confirmed these results. The same analysis and the results obtained by ion-exchange chromatography also provided evidence that the N-glycans of lamps are sialylated at 21 degrees C as much as at 37 degrees C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21 degrees C than at 37 degrees C. These results therefore indicate that incubation at 21 degrees C causes the lamps to reside longer within the Golgi complex, and such longer residence allows lamps to acquire more polylactosaminoglycan. These results also suggest that the time necessary for moving through the Golgi complex is a critical factor for poly-N-acetyllactosamine formation.
Wang
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WC
La Jolla Cancer Research Foundation, Cancer Research Center, California 92037.
Lee
N
N
Aoki
D
D
Fukuda
M N
MN
Fukuda
M
M
D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013696:Temperature; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D011134:Polysaccharides; C037199:poly-N-acetyllactosamine