Localization of epsilon-lysyl-gamma-glutamyl cross-links in alpha 2-macroglobulin-plasmin complex.
The major epsilon-lysyl-gamma-glutamyl cross-links present in the largely covalent 1:1 alpha 2-macroglobulin-(alpha 2M)-plasmin complex have been localized. Cross-linking engaged Lys607, -708, and -750 of the plasmin light chain, Lys550, -556, and -557 of the heavy chain-light chain connecting strand, Lys258 and -298 of the kringle 3 region, and Lys473 of the kringle 5 region of the plasmin heavy chain. Lys607, -708, and -750 accounted for 75% of all cross-linking, Lys550, -556, and -557 accounted for 20%, and Lys258, -298, and -473 accounted for 5%. Hence, cross-linking engaged only nine of the 41 Lys residues of plasmin, showing that, probably due to their large size, individual plasmin molecules become deposited in the large elongated binding cavity in alpha 2M in a relatively uniform way. The cross-linking of Lys residues in the heavy chain-light chain connecting strand to alpha 2M explains earlier findings that a substantial portion of the heavy chain is cross-linked to alpha 2M [Pizzo et al. (1986) Biol. Chem. Hoppe-Seyler 367, 1177-1182]. Although located in the heavy chain, Lys550, -556 and -557 should be considered part of the serine proteinase domain. That part of plasmin must be deeply buried in the alpha 2M structure in close vicinity to the four thiol esters, while most of the elongated heavy chain is protruding from the binding cavity. The pattern of cross-linked species seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that individual plasmin molecules are bound to alpha 2M through one or two cross-links. Bivalent cross-linking can take place within or between 360-kDa alpha 2M dimers, pointing to the tetrameric alpha 2M structure as the functional proteinase binding unit.
Jacobsen
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Department of Molecular Biology, University of Aarhus, Denmark.
Sottrup-Jensen
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D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism; D004151:Dipeptides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000378:metabolism*
D004151:Dipeptides; D010446:Peptide Fragments; D000511:alpha-Macroglobulins; C001299:epsilon-(gamma-glutamyl)-lysine; D002918:Chymotrypsin; D005341:Fibrinolysin