The poly-N-acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex

Wei Chun Wang, Ni Lee, Daisuke Aoki, Michiko N. Fukuda, Minoru Fukuda

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

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°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°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°C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37°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°C than incubation at 37°C. The analysis of oligosaccharides released by endo-β-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100% after incubation at 21°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°C as much as at 37°C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21°C than at 37°C. These results therefore indicate that incubation at 21°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.

Original languageEnglish
Pages (from-to)23185-23190
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number34
Publication statusPublished - 1991 Dec 5
Externally publishedYes

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Lysosome-Associated Membrane Glycoproteins
Golgi Apparatus
Association reactions
Glycoproteins
HL-60 Cells
Polysaccharides
Galactosidases
Selectins
Methylation
poly-N-acetyllactosamine
Endothelial cells
Ion Exchange Chromatography
Platelets
Chromatography
Electrophoresis
Oligosaccharides
Galactose
Sodium Dodecyl Sulfate
Methionine
Labeling

ASJC Scopus subject areas

  • Biochemistry

Cite this

The poly-N-acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex. / Wang, Wei Chun; Lee, Ni; Aoki, Daisuke; Fukuda, Michiko N.; Fukuda, Minoru.

In: Journal of Biological Chemistry, Vol. 266, No. 34, 05.12.1991, p. 23185-23190.

Research output: Contribution to journalArticle

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abstract = "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°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°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°C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37°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°C than incubation at 37°C. The analysis of oligosaccharides released by endo-β-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100{\%} after incubation at 21°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°C as much as at 37°C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21°C than at 37°C. These results therefore indicate that incubation at 21°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.",
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T1 - The poly-N-acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex

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AB - 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°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°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°C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37°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°C than incubation at 37°C. The analysis of oligosaccharides released by endo-β-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100% after incubation at 21°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°C as much as at 37°C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21°C than at 37°C. These results therefore indicate that incubation at 21°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.

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