Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction.
We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose. The occurrence of alpha 2M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-alpha 2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed alpha 2M subunits (94 and 180 kDa) and pMBP subunits (28kDa) in SDS-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing conditions. This band was also seen in eluates from the anti-alpha 2M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP-28 with alpha 2M.
Storgaard
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P
Department of Medical Microbiology, Odense University, Denmark.
Holm Nielsen
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E
Skriver
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Andersen
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Svehag
S E
SE
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002614:Chelating Agents; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017364:Collagenases / Q000494:pharmacology; D037222:Collectins; D007123:Immunoelectrophoresis, Two-Dimensional; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D011485:Protein Binding; D013552:Swine; D000511:alpha-Macroglobulins / Q000378:metabolism*
D002352:Carrier Proteins; D002614:Chelating Agents; D037222:Collectins; D000511:alpha-Macroglobulins; D017364:Collagenases