Interaction of Mannan Binding Lectin with α2 Macroglobulin via Exposed Oligomannose Glycans

Abstract

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor ␣ 2 macroglobulin (␣2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. ␣2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between ␣2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man 5-7 occupying Asn-846 on ␣2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active ␣2M (thiol ester intact) and proteasecleaved ␣2M (thiol ester cleaved). We demonstrate that MBL is able to interact with ␣2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to ␣2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral ␣2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.The protease inhibitor ␣ 2 macroglobulin (␣2M) 4 is a glycoprotein of 720 kDa, composed of four identical 180-kDa subunits, covalently linked in pairs by disulphide bridges. Each subunit has eight N-linked glycosylation sites located at Asn-32, Asn-47, Asn-224, Asn-373, Asn-387, Asn-846, Asn-968, and Asn-1401 (1) (see Fig. 1), a total of 32 N-linked glycosylation sites per ␣2M tetramer. ␣2M is an evolutionarily ancient protease inhibitor, which circulates in human plasma at 1-2 mg/ml and is found in a wide range of species from arthropods to mammals (2). There are two regions of major functional importance, a "bait region," which contains multiple cleavage sites for many different proteases (Fig. 1), and a second region, a reactive thiol ester (Fig. 1) also found in the complement components C3 and C4. ␣2M inhibits active proteases via a "trap mechanism" (3, 4), where cleavage of the "bait" region by the active protease triggers a conformational change in ␣2M (5) forming a cage-like structure around the protease. During the refolding, the protease or other "bystander" molecules (6) may become covalently bound via the thiol ester (7). The covalent binding, however, is not essential for protease inhibition (8). The entrapped protease is inhibited in its action on high molecular weight protein substrates, which can no longer access the cage. In contrast low molecular weight (sy...

Processing of CD109 by furin and its role in the regulation of TGF-β signaling

“…Furin (Furinase) is a major processing enzyme, mainly localized in the trans-Golgi network, and whose minimal specific sequence is Arg-X-(Lys/Arg)-Arg (Ouweland et al, 1990;Molloy et al, 1992;Rouille´et al, 1995;Steiner, 1998). Tunicamycin, a streptomycete antibiotic (Takatsuki et al, 1971), specifically inhibits the glycosylation of proteins at asparagine residues (Struck and Lennarz, 1977;Waechter and Harford, 1977;Guarnaccia et al, 1983;Arnold et al, 2006). A 1 mg/ml tunicamycin stock solution in 10 mM NaOH was prepared, and added to culture media at a final concentration of 5 mg/ml.…”

“…Tunicamycin completely inhibits mannose attachment on asparagine and is used for the detection of core proteins without Nlinked glycosylation (Struck and Lennarz, 1977;Waechter and Harford, 1977;Guarnaccia et al, 1983;Arnold et al, 2006). When 293-CD109 and R1273S cells were treated with 5 mg/ml tunicamycin for 24 h, a 155 kDa Role of furin-processed CD109 in TGF-b signaling S Hagiwara et al band was commonly detected using the anti-FLAG, anti-CD109-2 and anti-CD109-4 antibodies ( Figure 4a).…”

Surfactant Protein D Interacts with α2-Macroglobulin and Increases Its Innate Immune Potential

“…2B). A2M is present in the serum (1-2 mg/ml; 1.4 -2.8 M) (22,23) and the lungs (0.09 -2.02 g/ml; 0.13-2.8 nM) (18,19). Notably, the concentration of A2M drastically increases during lung infection, edema fluid accumulation, and inflammation (220 g/ml; 305.5 nM) (19).…”

“…The conformational change also renders the A2M to be recognized by the A2M receptor (LRP-1, CD91) (27,28). Each subunit of A2M has eight N-linked glycosylation sites that decorate the surface of the protein, and the serum collectin mannose-binding lectin recognizes the mannose residues present on A2M (22). However, its functional consequences are not clearly understood.…”

“…The bronchoalveolar lavage fluid (BALF) concentration of A2M can range between 0.03 and 8.12 g/ml in cystic fibrosis patients (20). Native A2M is a 720-kDa glycoprotein composed of four identical subunits linked as pairs by disulfide bonds (21)(22)(23). When A2M interacts with thiol, serine, aspartic acid, and metalloproteases, it allows cleavage of its "bait region" present within the tetrameric cage (24).…”