Carbohydrate-binding protein 35 (Mac-2), a laminin-binding lectin, forms functional dimers using cysteine 186

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Carbohydrate-binding protein 35 (CBP35), also known as macrophage surface antigen Mac-2, is a lectin with cell agglutination and laminin-binding properties.
• Previous research suggested CBP35 does not form multimers, despite dimerization potentially facilitating its functions.

Purpose of the Study:

• To investigate the multimeric forms of macrophage CBP35.
• To elucidate the biochemical mechanisms of CBP35 multimerization.
• To determine the role of dimerization in CBP35's interaction with laminin.

Main Methods:

• Purification of macrophage CBP35 using laminin affinity chromatography.
• Analysis of purified CBP35 under non-reducing conditions to identify species.
• Expression and purification of recombinant CBP35 (rCBP35) in E. coli.
• Site-directed mutagenesis to investigate the role of cysteine 186 in dimerization.
• Assessment of laminin binding affinity for monomeric and dimeric CBP35.

Main Results:

• Purified macrophage CBP35 exists as distinct species (Mr 35,000, 67,000, and 80,000) under non-reducing conditions.
• Recombinant CBP35 (rCBP35) forms disulfide-linked homodimers (Mr 67,000).
• Dimeric CBP35 exhibits higher affinity for laminin compared to monomeric form, via a lactosamine-dependent mechanism.
• Cysteine 186 is essential for CBP35 dimerization.

Conclusions:

• Macrophage CBP35 can exist in monomeric and dimeric forms, with dimerization mediated by disulfide bonds involving cysteine 186.
• Dimerization significantly enhances CBP35's binding to laminin, suggesting a role in cell-matrix interactions.
• Homo- and heterodimeric forms of CBP35 may contribute to its functions in cell-matrix interactions and growth regulation.