Influence of the mu-chain C-terminal sequence on polymerization of immunoglobulin M

Abstract

Immunoglobulin (IgM) is found in various states of covalent polymerization (microL)n, where n is typically 8, 10, or 12. The usual form of IgM of bony fish is tetrameric (8 microL units) as compared to the pentameric form (10 microL units) observed in cartilaginous fish and mammals. Two hypotheses were tested in this study. First, that the length of the mu-chain C terminus following Cys575 determines whether an IgM polymerizes as a tetramer or as a pentamer. This was tested by examining the covalent polymerization state of mouse IgM mutated to contain a series of mu-chain C-termini from bony and cartilaginous fish. The results proved this hypothesis wrong: mouse IgM bearing the C-terminal sequence of shark, salmon and cod mu-chain behaved identically to native mouse IgM, forming predominantly (microL)10 and (microL)12 forms. The second hypothesis was that an additional Cys residue near the C terminus of the mu-chain is responsible for the multiple covalent structures seen in IgM of the channel catfish. The addition of a catfish C terminus to the mouse mu-chain resulted, as predicted, in the production of a series of covalently bonded forms, with the major species being (microL)4. When a Ser-Cys unit was removed from the catfish C terminus added to the mouse mu-chain, this resulted in production of IgM indistinguishable in structure from that of wild-type mouse IgM.

Figure 1

Introduction of mutations into the 3′ coding region of the mouse μ gene. The Bam HI/KpnI fragment of the μ gene was the target for the introduction of mutations (shaded) into the Cμ4 exon. Following the confirmation of sequence the Bst EII/Kpn I fragment was cloned back into the parental Sp6(C414) and Sp6(S414) genes as shown. The asterisk (*) indicates Ser 414.

Figure 2

Analysis, by SDS–polyacrylamide slab gel electrophoresis under reducing conditions, of ³⁵S-labelled DNP-binding antibody produced by transfected and untransfected cell lines. X-10 is the parental cell line producing κ chains only, and Sp6 is the parental cell line producing both κ-and wild-type mouse μ-chains. Other lanes show the analysis of mutant mouse antibody produced by transfection of X-10 cells with a mouse μ gene in which the C-terminal sequence has been altered. The name of the species from which the introduced μ-chain C-terminal sequence was taken (Table 2) is indicated above the lanes. The presence of Cys or Ser at position 414 of the μ-chain is also indicated above each lane. The positions of standards (phosphorylase b, serum albumin, ovalbumin, carbonic anhydrase and trypsin inhibitor) are shown by arrows.

Figure 3

Analysis, by SDS–PAGE under non-reducing conditions, of ³⁵S-labelled DNP-binding antibody produced by transfected and untransfected cell lines. X-10 is the parental cell line producing κ-chains only, and Sp6 is the parental cell line producing both κ- and wild-type mouse μ-chains. Other lanes show the analysis of mutant mouse antibody, produced by transfection of X-10 cells with a mouse μ gene in which the C-terminal sequence has been altered. The name of the species from which the introduced μ-chain C-terminal sequence was taken (Table 2) is indicated above each lanes. The presence of Cys or Ser at position 414 of the μ-chain is also indicated above each lane. The mobility of the IgM standards [mouse IgM (TEPC 183 and Sp6) and catfish IgM] with eight, 10, or 12 ‘halfmer’ (μL) units is indicated by arrows.

Figure 4

Analysis, by SDS–PAGE under non-reducing conditions, of ³⁵S-labelled DNP-binding antibody produced by transfected and untransfected cell lines. X-10 is the parental cell line producing μ-chains only, and Sp6 is the parental cell line producing both κ- and wild-type mouse μ-chains. Other lanes show the analysis of mutant mouse antibody, produced by transfection of X-10 cells with a mouse μ gene in which the C-terminal sequence has been altered. The name of the species from which the introduced μ-chain C-terminal sequence was taken (Table 2) is indicated above the lanes. The mobilities of the IgM proteins used as standards are indicated by arrows and labelled according to the number of (μL) units they contain. The standards were mouse IgM [TEPC 183 and Sp6 with 10 and 12 (μL) subunits] and catfish IgM with two to eight (μL) subunits.