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Human monocyte-derived macrophages express an approximately 120-kD Ox-LDL binding protein with strong identity to
M A van der Kooij1, E M von der Mark, J K Kruijt
1Pharma Division, Hoffmann-La Roche Ltd, Basel, Switzerland.
Abstract:
A protein that specifically binds oxidized LDL (Ox-LDL) has recently been characterized in mouse peritoneal macrophages and identified as macrosialin, a protein with a molecular weight of 95 kD. First, the present work shows that human monocyte-derived macrophages express a membrane protein with a molecular weight of approximately 120 kD that selectively binds Ox-LDL. Second, we tested whether this approximately 120-kD Ox-LDL binding protein had any relation to CD68, the human homologue of macrosialin. The following evidence was obtained to support the role of CD68 as an Ox-LDL binding protein: (1) Ligand blots with Ox-LDL and Western blots with Ki-M6, an anti-human CD68 monoclonal antibody, revealed a single band with a molecular weight of approximately 120 kD under reducing and nonreducing condition. (2) The expression patterns of the approximately 120-kD Ox-LDL binding membrane protein and of CD68 paralleled each other during monocyte/macrophage differentiation. (3) Digestion with N-glycosidase F demonstrated that both CD68 and the Ox-LDL binding protein are glycoproteins; both showed a similar shift of approximately 18 kD in apparent molecular weight. (4) CD68, probed with monoclonal antibody Ki-M6, and the approximately 120-kD Ox-LDL binding protein were coprecipitated with EMB11, another anti-CD68 antibody. About 5000 molecules of CD68 are expressed on the cell surface of human macrophages. Ligation of 125I-Ki-M6 to cells leads to its internalization and degradation. This capacity would be sufficient to allow for the specific uptake and degradation of Ox-LDL. Taken together, these data support a role for CD68 as a specific Ox-LDL binding protein in human monocyte-derived macrophages.
Insights
Human macrophages express CD68, a protein that binds oxidized low-density lipoprotein (Ox-LDL). This finding suggests CD68 plays a key role in the uptake and degradation of Ox-LDL, potentially impacting cardiovascular health.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Oxidized low-density lipoprotein (Ox-LDL) is implicated in atherosclerosis.
- Macrosialin, a 95 kD protein in mouse macrophages, binds Ox-LDL.
- The human homologue of macrosialin is CD68.
Purpose of the Study:
- To investigate if human macrophages express a protein that binds Ox-LDL.
- To determine if CD68 is the Ox-LDL binding protein in human macrophages.
Main Methods:
- Ligand blotting and Western blotting using anti-CD68 antibody (Ki-M6) and Ox-LDL.
- Analysis of CD68 and Ox-LDL binding protein expression during monocyte/macrophage differentiation.
- N-glycosidase F digestion to assess glycoprotein nature.
- Coprecipitation assays with anti-CD68 antibodies (Ki-M6 and EMB11).
Main Results:
- Human macrophages express a ~120 kD membrane protein that binds Ox-LDL.
- This protein shares characteristics with CD68, including molecular weight and expression patterns during differentiation.
- Both CD68 and the Ox-LDL binding protein are glycoproteins.
- CD68 and the Ox-LDL binding protein were coprecipitated, confirming their association.
Conclusions:
- CD68 functions as a specific Ox-LDL binding protein in human monocyte-derived macrophages.
- The cell surface expression and internalization capacity of CD68 support its role in Ox-LDL uptake and degradation.