Light Chain Sequences of Human IgM Cold Agglutinins

The variable or V regions of the Ig heavy chains have invariable, variable, and hypervariable sections (1, 2). The individual antigenic specificities or idiotypes (3, 4) have their localization in the hypervariable regions or near these regions (5). In the relatively invariant sections there are certain positions in the amino acid sequence, which permit the classification of the heavy chains of different myeloma proteins into three main heavy-chain variable region (V~) subgroups. However, because of the difficulties in sequencing multiple proteins, only limited data are available on the incidence and distribution of these three different types. The aim of the present study was to establish serological typing systems for the determination of VH subgroups in myeloma proteins and in the Ig of normal sera. Such a system would have many advantages in addition to procedural simplification; characterization of the membrane Igs of lymphocytes would be a specific example. The typing systems developed in this study utilized antisera made against myeloma proteins with known Vn subgroups as determined by amino acid sequence analysis. Certain of these antisera after absorption could be made specific for each of the three subgroups. Materials and MethodsMyeloma Proteins. Myeloma proteins with known amino acid sequence of the V region of the heavy chain were from our own laboratories or received from other sources. Some of the myeloma proteins used were kindly provided by Doctors H. Bennich, D. Capra, M. Fougereau, and F. Putman. The proteins with known VH subgroup based on amino acid sequence are given in Table II; many of these have been previously described (2,(6)(7)(8)(9)(10). A number of other proteins had only been partially typed by testing for blocked or unblocked N-terminal residues (11) or with a recently developed chemical typing system based on peptide analysis (12). In addition, a large number of myeloma proteins with known Ig class, subclass, and light-chain type but without known VH subgroup were tested.Myeloma proteins were initially isolated from the whole sera by starch block electrophoresis (13,14) or by DEAE-cellulose (Whatman DE-52) column chromatography in 0.015 M phosphate buffer, pH 7.6 (14--16). In other cases the myeloma proteins were eluted from a DEAE-cellulose column by a gradient from 0.015 M to 0.3 M phosphate buffer, pH 7.6. The myeloma proteins obtained were finally gel filtrated on a Sephadex G150 column under neutral conditions to give the IgG, IgA, IgM, IgD, or IgE proteins used. The purity of the various fractions of myeloma proteins were tested by agarose gel electrophoresis aider the DEAE-cellulose and G150 steps.

Section: Discussionmentioning

confidence: 99%

Serological detection of variable region (Vh) subgroups of Ig heavy chains.

Førre¹,

Natvig²,

Kunkel³

1976

“…Perhaps the most pertinent to the present study, since they arose in genetically different individuals, are the human monoclonal cold agglutinins and antigamma globulins studied by Kunkel, Capra, and their colleagues (44,45) . Though no data on heavy chain structure have been presented, amino acid sequence analysis of the light chains from 6 of 10 cold agglutinins showed minimal differences among them (46) . Likewise, antigamma globulins selected for similarity in shared idiotypic determinants exhibit remarkable structural similarities in either their light or their heavy chains (47) .…”

Section: Discussionmentioning

confidence: 99%

Structural, functional, and idiotypic characteristics of a phosphorylcholine-binding IgA myeloma protein of C57BL/ka allotype.

Claflin¹,

Rudikoff²,

Potter³

et al. 1975

An IgA phosphorylcholine (PC)-binding myeloma protein with IgCH allotypic determinants different from those of BALB/c mice is characterized. The myeloma, CBPC 2, was induced in the CB-20 strain of mice which is congenic to BALB/c but differs from it by carrying the A15 allotypic determinant of C57BL/ka mice. Sequence analysis of the CBPC 2 light chain through the first hypervariable region, as well as isoelectric point analysis, show that this chain is indistinguishable from that of T15, a PC-binding myeloma protein of BALB/c origin. The heavy chains of CBPC 2 and T15 differ by only two amino acids (positions 14 and 16) through the first hypervariable region. As measured by inhibition of precipitation, both CBPC 2 and T15 have the same specificity for PC, glycerophosphorylcholine, acetylcholine, and choline. In addition, CBPC 2 possesses the binding site-associated idiotypic determinant which is present on T15. However, like normal or induced C57BL/6 anti-PC antibody, it does not possess the nonbinding site idiotypic determinant.

“…Thus, in some, but not all strains of mice, antibodies produced to a-l,3-1inked dextrans (27)(28) or PC (6, 7) exhibit cross-idiotypic specificity and in the former response, the idiotypic specificity appears to be site related (29). Cross-idiotypic specificity also exists within groups of myeloma proteins with similar specificities (30)(31)(32), and in some of these systems as well as others, structure-function (33)(34)(35) or idiotype-function (29,36) relationships are currently being delineated. Among the human IgM proteins with anti-'g-globulin activity which have been extensively studied by Kunkel and his colleagues, two major groups appear which are classified on the basis of shared idiotypic determinants (32).…”

Section: Extent Of Shared Idiotypy On Hopc 8 and Mouse Anti-pc Antibomentioning

confidence: 99%

Clonal Nature of the Immune Response to Phosphorylcholine

Claflin

Davie

1974

102

Studies of the binding characteristics for a number of choline analogues have revealed that antiphosphorylcholine (PC) 1 antibodies from 15 different inbred mouse strains which differ in histocompatibility and heavy-chain allotype are remarkably restricted and show the same binding specificity as a PC-binding myeloma protein, HOPC 8. 2' 3 This suggests that the anti-PC antibodies of all mice possess similar binding sites for PC. Indeed Cosenza and KShler (5) and others (2, 6, 7) using mouse antimouse idiotypic antisera have dearly demonstrated that anti-PC antibody from BALB/c possesses antigenic determinants shared by HOPC 8. Paradoxically however, antibodies from other strains, such as C57BL/6, A, and CE mice (2, 5-7), do not have this idiotypic specificity even though their antibody specificity cannot be distinguished from that of BALB/c. These studies indicate that variable-region determinants differ in the anti-PC antibodies of different strains of mice, but since the hapten inhibition of binding to idiotype was not studied, it is not dear that these idiotypic differences lie in the binding region. This consideration is crucial to the analysis of antibody combining-site diversity since selective pressures exerted on binding site and nonbinding site regions may be different.