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Regulated expression of a 16-kd galectin-like protein in activated rat macrophages
G Rabinovich1, L Castagna, C Landa
1Departamentos de Bioquimica Clinica y Quimica Biológica, Facultad de Ciencias Quimicas, Universidad Nacional de Córdoba, Argentina.
Insights
Researchers identified a galectin-like protein in rat macrophages, finding its expression increases with inflammation and activation. This protein is present in the cytoplasm and on the cell surface.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Galectins are a family of carbohydrate-binding proteins involved in various cellular functions.
- The presence and role of specific galectin-like proteins in rat mononuclear cells, particularly macrophages, require further investigation.
Purpose of the Study:
- To investigate the presence and characteristics of a galectin-like protein in rat mononuclear cells.
- To determine the subcellular localization and expression patterns of this protein.
- To explore how inflammatory stimuli modulate the expression and localization of this galectin-like protein.
Main Methods:
- Utilized a polyclonal antibody against chicken liver lectin for immunochemical detection.
- Performed Western blot assays on various rat cell fractions (spleen, T cell-enriched, B cell/macrophage-enriched, peritoneal).
- Conducted immunocytochemistry, flow cytometry (ED1 marker), hemagglutination assays, and lactosyl-Sepharose chromatography.
Main Results:
- A ~16 kDa galectin-like protein was detected constitutively in rat mononuclear cells, primarily in macrophages (M phi).
- The protein is localized in both the cytoplasmic compartment and associated with the cell surface.
- Expression was significantly upregulated in macrophages treated with phorbol ester and formylmethionine oligopeptide, involving protein kinase C activation.
Conclusions:
- Rat macrophages express a cell-surface and cytoplasmic galectin-like protein.
- This protein's expression and localization are modulated by inflammatory stimuli and protein kinase C activation.
- The findings suggest a role for this galectin-like protein in macrophage function and inflammatory responses.
Abstract:
We investigated the presence of a galectin-like protein in rat mononuclear cells using a polyclonal antibody raised against a soluble lactose-binding lectin purified from adult chicken liver that immunoreacted strongly with a broad protein band of about 16 kd in Western blot assays. Immunochemical studies revealed a constitutive expression of this protein in mononuclear cells mainly in the macrophage (M phi) population. Subcellular localization was assessed by Western blot assays of the cytosolic and membrane fractions of different cell populations studied: (1) spleen mononuclear cells, (2) T cell-enriched, (3) B cell- and M phi-enriched populations, and (4) peritoneal cells, processed in the presence of lactose. In broad agreement with immunocytochemical studies of nonpermeabilized and permeabilized cells, Western blot assays suggest that this protein is localized mainly in the cytoplasmic compartment but also associated with the cell surface. By flow cytometric analyses we detected about a 14% of ED1 double-positive cells corresponding to macrophages that constitutively express this galectin-like protein associated with their cell surface. The cytosolic fraction obtained from the M phi-enriched cell population showed hemagglutinating activity specifically inhibited by beta-galactoside-related sugars. Moreover, this galectin-like protein was retained in a lactosyl-Sepharose matrix and specifically eluted with lactose. In this work, evidence is also provided to show that different stimuli are able to modulate the expression of the galectin-like protein. Expression was upregulated in inflammatory and activated macrophages, revealing a significant increase in phorbol ester- and formylmethionine oligopeptide-treated cells. Both stimuli involving protein kinase C activation pathway have been able not only to up-regulate the total expression of this protein but also to modulate its subcellular localization.
