Functional studies on recombinant domains of Mac-2-binding protein.
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.
Hellstern
Simon
S
Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Sasaki
Takako
T
Fauser
Charlotte
C
Lustig
Ariel
A
Timpl
Rupert
R
Engel
Jürgen
J
D000951:Antigens, Neoplasm; D001483:Base Sequence; D001665:Binding Sites; D014408:Biomarkers, Tumor; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D002942:Circular Dichroism; D017931:DNA Primers; D019281:Dimerization; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D016326:Extracellular Matrix Proteins
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D017931:DNA Primers; D006023:Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014415:Tunicamycin; C081856:LGALS3BP protein, human; D016326:Extracellular Matrix Proteins