Domain-switched mouse IgM/IgG2b hybrids indicate individual roles for C mu 2, C mu 3, and C mu 4 domains in the regulation of the interaction of IgM with complement C1q.
Although polymeric IgM and monomeric IgG are potent activators of the classical complement pathway, previous studies have indicated that monomeric IgM is inactive. To understand this and to examine the roles of the individual mu domains in complement activation, we created a set of IgM/IgG2b mouse chimeric Abs in which homologous domains of both Abs have been interchanged, either singly or together with adjacent domains. The monomer subunits (H2L2) of the resulting chimeras were analyzed for their capacities to bind C1q and to initiate complement-mediated lysis (CML) of haptenated erythrocytes. When C gamma 2 was flanked by C mu 4, the inherent C1q-binding activity of the C gamma 2 domain was lost. This demonstrates that C mu 4 can suppress the C1q-binding activity of the adjacent C gamma 2 domain, and suggests that C mu 4 may exert a similar effect on the C mu 3 domain in the IgM monomer subunit. When C mu 3 was located in an IgG2b background and potentially freed from the constraints imposed by the IgM background, the monomer was not able to bind C1q or initiate CML. This suggests that these activities are not expressed inherently in the C mu 3 domain. The transplantation of C mu 3 together with C mu 4 into the IgG background permitted polymer formation. This polymer was able to bind C1q, although neither the monomer nor the polymer forms were active in CML; conversely, all IgM polymers with a transplanted C gamma 2 domain were active in both C1q binding and CML, and demonstrated apparent Kd values similar to that of wild-type IgM.
Chen
F H
FH
Department of Biochemistry, University of Toronto, Ontario, Canada.
Arya
S K
SK
Rinfret
A
A
Isenman
D E
DE
Shulman
M J
MJ
Painter
R H
RH
D000818:Animals; D015922:Complement C1q / Q000378:metabolism*; D003171:Complement Pathway, Classical / Q000276:immunology; D005822:Genetic Vectors / Q000276:immunology; D006461:Hemolysis / Q000276:immunology; D007127:Immunoglobulin Constant Regions / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007075:Immunoglobulin M / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D010954:Plasmacytoma; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000502:physiology; D014162:Transfection / Q000276:immunology; D014407:Tumor Cells, Cultured
D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins; D015922:Complement C1q