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Functional studies on recombinant domains of Mac-2-binding protein

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons78606

Sasaki,  T.
Former Research Groups, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78797

Timpl,  R.
Former Research Groups, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Hellstern, S., Sasaki, T., Fauser, C., Lustig, A., Timpl, R., & Engel, J. (2002). Functional studies on recombinant domains of Mac-2-binding protein. Journal of Biological Chemistry, 277(18), 15690-15696.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-6F3A-6
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.