Abstract
Mac-2-binding protein (M2BP) is a secreted glycoprotein suggested to have a role in host defense. It forms linear and ring-shaped oligomers, with each ring segment being composed of two monomers. We have produced recombinant human M2BP fragments comprising domains 1 and 2 (M2BP-1,2) and domains 3 and 4 (M2BP-3,4) in 293 human kidney cells to characterize structural and functional properties of M2BP. Both fragments were obtained in a native and glycosylated form, as analyzed by CD spectroscopy, trypsin susceptibility, and enzymatic deglycosylation. These results strongly suggest that both fragments are autonomous folding units. All three potential N-glycosylation sites in M2BP-1,2 and all four in M2BP-3,4 were found to be occupied. M2BP-1,2 expressed in tunicamycin-treated cells contained no glycosyl residues, indicating that O-glycosylation is not occurring. Ultracentrifugation revealed that M2BP-1,2 is homogeneously dimeric in the nanomolar range reflecting the properties of intact M2BP. Domain 2 (BTB/POZ domain) is thus identified as the dimerization domain of M2BP, because it has been formerly shown that recombinant domain 1 is monomeric. M2BP-3,4 showed a concentration-dependent self-association, and aggregates of different size and shape were shown by electron microscopy. In contrast to this irregular aggregation of M2BP-3,4, it has been formerly shown that a fragment comprising domains 2-4 still has the ability to form ring-like structures, although the rings are protein-filled, and thus domain 2 appears to be indispensable for ring formation. Solid phase assays showed that M2BP-3,4 contains binding sites for galectin-3, nidogen, and collagens V and VI, whereas M2BP-1,2 is inactive in binding. Both fragments showed no cell adhesive activity in contrast to native M2BP, suggesting that a concerted binding action and/or multivalent interactions of rings are necessary for cell attachment.
Highlights
Mac-2-binding protein (M2BP)[1] is a secreted glycoprotein present in the extracellular matrix of several tissues (1) and in extracellular fluids such as serum and milk (2)
In contrast to this irregular aggregation of M2BP-3,4, it has been formerly shown that a fragment comprising domains 2– 4 still has the ability to form ringlike structures, the rings are protein-filled, and domain 2 appears to be indispensable for ring formation
M2BP-1,2 and M2BP-3,4: Expression and Investigation of the Oligosaccharide Moieties—Fragments consisting of domains 1 and 2 (M2BP-1,2; residues 19 –254) and of domains 3 and 4 (M2BP-3,4; residues 250 –585) were prepared according to the predicted domain boundaries for human M2BP (11). cDNAs were produced by PCR amplification to code for a Nterminal His[6] tag
Summary
Mac-2-binding protein (M2BP)[1] is a secreted glycoprotein present in the extracellular matrix of several tissues (1) and in extracellular fluids such as serum and milk (2). Recombinant domain 1 (M2BP-1) is monomeric and is inactive in binding to extracellular matrix ligands and in cell attachment. A fragment consisting of putative domains 2– 4 (M2BP-2,3,4) aggregates to heterogeneous, protein-filled ring-like structures and retains the potential for binding to extracellular matrix ligands and for cell adhesion (11). In the present study we have produced recombinant M2BP fragments consisting of domains 1 and 2 (M2BP-1,2) and domains 3 and 4 (M2BP-3,4) to further characterize the function of the different domains. Both fragments were obtained in native and glycosylated forms, suggesting that they are autonomous folding units.
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