Functional Studies on Recombinant Domains of Mac-2-binding Protein

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 ringlike 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. 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). Elevated levels are observed in some tumors and viral infections (3, 4). In vitro, human M2BP induces production of interleukin-1, interleukin-6, and other cytokines by blood monocytes and stimulates natural killer cell and lymphokine-activated killer cell activity (5, 6). Mouse cyclophilin C-associated protein is 69% identical to M2BP, and it seems likely that the two proteins are functional homologues. Gene-targeted cyclophilin C-associated protein-deficient mice are viable but show an up-regulation of the endotoxin and proinflammatory response (7).M2BP is extensively glycosylated and interacts with galectin-3 (former name Mac-2), and it also interacts with other extracellular proteins such as collagens IV, V, and VI, fibronectin, and nidogen (1, 2, 8, 9). M2BP binds to galectin-3 on the cell surface and induces homotypic cell aggregation (10). ␤ 1 -Integrin-mediated cell adhesion to M2BP was also demonstrated (1).Tissue extracted and recombinant M2BP for...

Human Galectin 3 Binding Protein Interacts with Recombinant Adeno-Associated Virus Type 6

“…G3BP is a highly glycosylated glycoprotein with all seven potential N-glycosylation sites occupied by complex oligosaccharide chains containing sialic acid (21) but not heparan sulfate (42). The mean concentration of this protein in the plasma of normal children (n ϭ 10) is 6.3 g/ml (19).…”

“…Only rAAV-6 and rAAV-1(E531K) were precipitated under such conditions. linkage of different components and for an increase in binding activity (21,31).…”

“…Other studies carried out on 100 adults have shown that normal concentrations range from 1.3 to 17 g/ml (enzyme immunoassay for the quantitative determination of s90K/Mac-2BP in cell culture supernatant; human serum and plasma package insert; IBL International GMBH, Hamburg, Germany; http: //www.ibl-international.com/magento/media/catalog/product/B /E/BE59271_IFU_en_s90_MAC-2BP_ELISA_V2011_04_sym2 .pdf). From a structural point of view, G3BP is composed of several domains consisting of a single scavenger receptor cysteine-rich (SRCR) domain at the N terminus, two central domains related to the dimerization domains BTP/POZ and IVR of the Drosophila kelch protein, and a C-terminal domain (21,23,31,42). The evolutionarily conserved SRCR domain is found in diverse transmembrane and secreted glycoproteins, including CD5, CD6, M130, complement factor 1, and WC1 antigen (41).…”

The Conserved Scavenger Receptor Cysteine-Rich Superfamily in Therapy and Diagnosis

“…Each subunit of approximately 90 kDa possesses four distinct domains: an Nterminal group A SRCR domain; a putative member of the BTB/POZ (broad complex, tramtrack and bric-abrac/poxvirus and zinc finger) dimerization domains; an IVR (intervening region) related to the kelch protein from D. melanogaster (Robinson and Cooley 1997;Mü ller et al, 1999); and a C-terminal region with no similarity to any other known protein (Mü ller et al, 1999). The BTB/POZ and IVR domains are responsible for protein oligomerization, as determined by studies with recombinant truncated protein forms (Hellstern et al, 2002). The protein can be found only in monomeric form under strongly denaturing conditions.…”

Involvement of NF-κB-mediated expression of galectin-3-binding protein in TNF-α-induced breast cancer cell adhesion

“…G3BP is present in the extracellular matrix of several tissues (16), tumor cell medium, milk, and serum; and it was shown to be a non-covalently linked oligomer with a molecular mass of >1000 kDa (17,18). Northern blotting and immunohistochemical analyses have revealed elevated expression of G3BP in pancreatic, breast and lung cancer (19)(20)(21).…”