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Depletion and Reconstitution of Macrophages in Mice
Published on: August 1, 2012
Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal
1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Abstract:
Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of D-[2-3H]mannose, D-[6-3H]galactose, N-acetyl-D-[1-3H]glucosamine and L-[5,6-3H]fucose by 2-15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.
Insights
Phagocytosis of zymosan by macrophages alters macrosialin glycosylation, impacting its structure and lectin binding. This highlights differential glycosylation
Area of Science:
- Immunology
- Cell Biology
- Glycobiology
Background:
- Macrosialin (mouse CD68) is a macrophage lysosomal protein with N-linked glycosylation and a sialylated domain.
- Glycosylation patterns are crucial for protein function and cellular processes.
Purpose of the Study:
- To investigate how phagocytosis affects macrosialin glycosylation in macrophages.
- To understand the role of differential glycosylation in macrophage biology.
Main Methods:
- Inflammatory peritoneal macrophages were treated with zymosan to induce phagocytosis.
- Incorporation of radiolabeled sugars (mannose, galactose, glucosamine, fucose) was measured.
- Lectin binding assays were performed to assess changes in glycan structures.
- N-Glycanase digestion was used to analyze N-linked glycans.
Main Results:
- Phagocytosis of zymosan significantly altered macrosialin's N-linked and O-linked glycosylation.
- Specific sugar incorporation increased, indicating active glycan processing.
- Changes in glycosylation affected macrosialin's binding affinity to various lectins.
- N-Glycanase digestion revealed distinct alterations in N-linked glycan structures.
Conclusions:
- Phagocytosis induces specific glycoform changes in macrosialin, dependent on particle internalization.
- Differential glycosylation plays a key role in the biology of macrosialin, phagosomes, and macrophages.
- These findings offer insights into macrophage functional regulation via glycosylation.
