Structural and idiotypic characterization of the L chains of human IgM autoantibodies with different specificities

We have determined the V region amino acid sequence and/or serologic markers (kIIIb, PSL2, and PSL3) of 24 IgM monoclonal autoantibodies with specificities of anti-gamma-globulin (RF), anti-I (cold agglutinin), anti-low density lipoprotein and anti-intermediate filaments. The data emphasize the overwhelming selection of the HumKv325/VkIIIb L chain for this family of autoantibodies. The few amino acid substitutions found within the VL regions were mainly concentrated in the complementarity-determining region 1. JK and CK genes did not show the same pattern of restriction. There is a good correlation between the amino acid sequence and the presence of the kIIIb marker. The idiotypic marker PSL2 was present in 34 out of 35 kIIIb L chains analyzed (97%) and the PSL3 in 27 (80%). Moreover, the hydrophilicity and antigenic profiles of these L chains corroborate the presence of the epitopes detected by the anti-CRI. These results demonstrate a restricted selection of the Vk genes used by a family of self reacting proteins, and an unusual evolutionary conservation of the idiotypic structure that may be involved in the network regulation.     

Autoantibody-encoding kappa L chain genes frequently rearranged in lambda L chain-expressing chronic lymphocytic leukemia

Patients with kappa L chain expressing chronic lymphocytic leukemia (CLL) frequently have leukemia cells reactive with a murine mAb, designated 17.109. Raised against a monoclonal IgM rheumatoid factor autoantibody, this mAb recognizes a major kappa-L chain-associated cross reactive Id, designated 17.109-CRI. Molecular studies reveal that the 17.109-CRI in CLL is a serologic marker for expression of a conserved kappa L chain V region gene (V Kappa gene) of the V Kappa 3 subgroup, designated Humkv325. We isolated an upstream gene fragment of Humkv325 to examine for Ig gene rearrangements of this and other closely related V Kappa 3 genes by Southern analyses. Consistent with Humkv325 encoding the 17.109-CRI, we find that the genomic DNA from all 17.109-reactive leukemia cell populations have gene rearrangements that are detected using this probe. In addition, we observe V Kappa 3 gene rearrangements frequently in the genomic DNA of lambda L chain-expressing leukemia cells. Of the genomic DNA from 33 lambda-L chain-expressing CLL samples, 8 (24%) had additional nongerm-line bands detected with the Humkv325 probe. Consistent with these bands representing Ig gene rearrangements, the additional band in each but one sample also hybridized with probes specific for the J Kappa region and/or the kappa-deleting element. Using the polymerase chain reaction (PCR), we examined the genomic DNA from all lambda L chain-expressing CLL for V Kappa 3 gene rearrangements to J Kappa and/or Kde. PCR on each DNA sample with V Kappa 3 gene rearrangements detected by Southern analysis generated gene fragments that hybridized specifically with oligonucleotides corresponding to framework or CDR of the Humkv325 gene. Nucleic acid sequence analyses of representative samples confirmed that these DNA contained abortive Humkv325 gene rearrangements. PCR for rearranged V Kappa 3 genes in the DNA of other lambda-L chain-expressing CLL either did not generate any PCR product or produced fragments that failed to hybridize with all Humkv325 oligonucleotide probes. Nucleic acid sequence analyses of the latter demonstrated that these represent abortive V Kappa gene rearrangements involving another conserved V Kappa 3 gene, designated Vg. These studies indicate that Humkv325 and Vg frequently may undergo Ig gene rearrangement independent of their expression. As such, the frequent use of Humkv325 in CLL may be secondary, in part, to an enhanced propensity of this V Kappa 3 gene to undergo genetic rearrangement during B cell ontogeny.     

Dissociation of expression of two rheumatoid factor cross-reactive kappa L chain idiotopes in rheumatoid arthritis.

mAb 6B6.6 and 17.109 recognize two distinct kappa III L chain cross-reactive idiotopes (CRI) present on approximately 2/3 of IgM kappa rheumatoid factor (RF) paraproteins. To determine the distribution of these two CRI and their relationship to each other among polyclonal RF, sera from 86 RA patients and 49 controls were analyzed for the presence of 6B6.6- and 17.019-bearing RF by using sensitive solid phase ELISA. Levels of CRI(+) RF were estimated by using 6B6.6(+) and 17.019(+) RF standards. Detectable levels (greater than or equal to 195 ng/ml) of CRI(+) RF were rarely present in the control sera (8% for 6B6.6; 0% for 17.109), whereas 59% of RA sera contained measurable CRI(+) RF (48% for 6B6.6; 35% for 17.109; 21% for both). Where detected, CRI(+) RF were present in low concentrations (6B6.6: 1.21 +/- 1.56 micrograms/ml; 17.109: 1.20 +/- 1.15 micrograms/ml) and constituted a small fraction of the total IgM RF in these sera (6B6.6: 0.9 +/- 2.2%; 17.109: 0.8 +/- 0.9%). There was no correlation between either RF CRI and levels of IgM RF (r less than 0.1, p greater than 0.5). Levels of 6B6.6(+) RF did not correlate with 17.109(+) RF (r = -0.11, p = 0.47). In selected sera that contained both RF CRI, it was possible to selectively absorb 6B6.6(+) RF. Taken together, these data indicate the mutual independence of these two RF CRI among polyclonal RF and suggest the presence of distinct regulatory mechanisms governing their expression. Moreover, that these two CRI constitute a small proportion of polyclonal RF, in contrast to their striking predominance among monoclonal RF paraproteins, argues for the importance of other germline VL genes contributing to polyclonal RF production or the presence of extensive somatic mutation among polyclonal RF in RA.     

Complete amino acid sequence of the heavy-chain variable region from an A/J mouse antigen-nonbinding monoclonal antibody bearing the predominant arsonate idiotype

The 1F6 hybridoma protein, exhibiting the predominant cross-reactive idiotype (CRI) associated with the immune response to p-azophenylarsonate in A/J mice but failing to bind the hapten arsonate, was elicited following immunization with rat anti-CRI [Wysocki, L.J., & Sato, V. (1981) Eur. J. Immunol. 11, 832-839]. The dissociation of idiotype and antigen binding in this hybridoma provides an opportunity to determine structural features involved in antigen binding and idiotypic sites. The complete heavy-chain variable region (VH) amino acid sequence was obtained by automated Edman degradation of the intact chain and fragments due to CNBr cleavage, trypsin digestion, mild acid hydrolysis, and carboxypeptidase A digestion of a CNBr fragment. Comparison of the CRI+ arsonate-nonbinding 1F6 sequence with the CRI+ germ-line VH gene sequence reveals that the 1F6 heavy chain differs from the germ-line-encoded amino acid sequence at seven positions within VH [Siekevitz, M., Gefter, M. L., Brodeur, P., Riblet, R., & Marshak-Rothstein, A. (1982) Eur. J. Immunol. 12, 1023-1032]. The 1F6 VH appears to arise from the CRI+ germ-line VH by somatic mutation at at least seven amino acid residues, each of which could be due to a single nucleotide base change. The diversity (D) gene-encoded segment of 1F6 is similar to that of the CRI+ antigen-binding hybridoma 36-65 except for two amino acid substitutions. Further, the idiotype (CRI) is preserved despite use of a JH4 gene segment in 1F6 as compared to JH2 in all CRI+ arsonate-binding hybridomas examined to date.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

Genetic analysis of human B cell hybridomas expressing a cross-reactive idiotype

We have examined the genetic basis for the expression of a human cross-reactive idiotype (CRI) commonly found on monoclonal IgM rheumatoid factors. The CRI was identified with a monoclonal antibody (17.109) and has been localized previously to the kappa-variable region. By using the human lymphoblastoid cell line WI-L2-729-HF2, and mononuclear cells from several sources, a panel of hybridomas was generated that produced 17.109 CRI-positive Ig. A recently cloned human germ-line V kappa III gene, Humkv305, served as a probe to identify genes which were rearranged and expressed in 17.109 CRI-positive and -negative hybridomas. This probe, when hybridized to human genomic DNA under stringent conditions, identified only two to five germ-line bands. In 10 separate 17.109 CRI-positive hybridoma clones, an additional rearranged V kappa band was identified. The probe did not anneal to rearranged V kappa bands in hybridoma clones that produced kappa-chains lacking the CRI. RNA dot-blot studies provided evidence for expression of genes hybridizing to the Humkv305 probe. The results indicate that the 17.109 CRI is a serologic marker for a single V kappa gene, or a small family of closely related V kappa genes, which is identified by the Humkv305 probe.     

VH and VL gene usage by murine IgG antibodies that bind autologous insulin

To assess the recognition structures of antibodies that bind a self-Ag, we used mRNA analysis to identify the V region genes of IgG antibodies that bind autologous insulin. Four anti-insulin mAb from primary immunization of BALB/c mice use different combinations of H and L chain V region genes. Two VH genes are from the V-gam 3-2 and V-gam 3-8 families that are infrequently expressed in adult BALB/c mice, and two VH genes are members of the J558 family. Each anti-insulin antibody uses a different Vk gene family. Two antibodies express common Vk genes (Ox1 and Vk21C), whereas two other Vk genes are unusual in BALB/c mice. One Vk gene may represent a BALB/c equivalent of the VkOx2 subfamily and another is identical to a Vk used by anti-idiotypic antibodies from C57Bl/6 mice. When compared with known germ-line counterparts, all of the Vk sequences are close to germ-line configuration. In contrast, the germ-line counterparts for the anti-insulin VH genes are not known, however, they differ only in five to seven predicted amino acids from VH of other expressed antibodies. One antibody (mAb 123) differs in one amino acid in complementarity-determining regions 1 and 2 from the VH of the murine tumor BCL1, and another (mAb 126) employs an unmutated DFL16.1 germ-line D segment. These data suggest that antibodies binding autologous insulin use V gene components that are not extensively mutated, even when derived by immunization with heterologous insulin.     

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.     

Incidence of three cross-reactive idiotypes on human rheumatoid factor paraproteins

The basis for rheumatoid factor (RF) production in autoimmune or lymphoproliferative diseases cannot be understood without defining the molecular factors that dictate RF structure and specificity. Recently three different mAb (6B6.6, 17.109, and G6) have been developed that define cross-reactive idiotypes (CRI) on intact L or H chains of human monoclonal RF cryoglobulins. However, the true incidence of these CRI among RF and their relationship to each other have not been delineated. In the present experiments, a panel of 163 randomly selected IgM paraproteins was evaluated for the expression of the two kappa L chain CRI, 6B6.6 and 17.109, and the H chain CRI, G6. Among the paraproteins with kappa L chains, 14% expressed the 17.109 CRI, and 9% expressed the 6B6.6 CRI. Both ELISA and Western immunoblotting experiments showed that the two L chain CRI were mutually exclusive. Anti-IgG activity was documented in 22 of the IgM-kappa paraproteins, among which mAb 6B6.6 reacted with 7 (32%) and mAb 17.109 with 6 (27%). Both CRI were expressed exclusively by L chains within the kappaIII variable gene subgroup. Although 17.109 CRI+ paraproteins had kappaIIIb L chains, none of the 6B6.6 CRI+ paraproteins possessed L chains with this kappa sub-subgroup specific Ag. The G6 CRI was found predominantly among RF paraproteins and was frequently yet not exclusively associated with the 17.109 CRI+ L chains. Additional experiments were performed on a panel of normal adult human sera and documented the presence of 6B6.6 and 17.109 CRI on a small percentage (0.1 to 2.0%) of IgM from most individuals. These data indicate that 1) the mAb 6B6.6 and 17.109 identify two major and distinct CRI among IgM-RF paraproteins, 2) both CRI are associated exclusively with kappaIII L chains, 3) kappaIIIb and kappaIII non-b L chains are equally prevalent among IgM-RF, 4) the G6 H chain CRI is frequently associated with 17.109 CRI+ L chains, but not with 6B6.6 CRI+ L chains, and 5) although the ability to make 6B6.6 and 17.109 CRI+ kappa L chains is common in humans, these CRI are present in low concentrations in normal IgM.     

Sequence similarities and cross-idiotypic specificity of L chains among human monoclonal IgM kappa with anti-gamma-globulin activity

The complete amino acid sequence of five light chain variable (V) regions of human monoclonal IgM kappa rheumatoid factors (RF) was determined, and their cross-reactive idiotypes (CRI) were characterized with antibodies induced by immunization with synthetic peptides PSL2 and PSL3, corresponding to the second and third complementarity-determining regions (CDR) of the SIE light chain. Together with two additional RF studied previously, all seven RF belong to the V kappa IIIb sub-subgroup. The region encoded by the V kappa gene segment (positions 1 to 95) in all seven proteins was virtually identical in primary structure, whereas the sequence from positions 96 to 108 defined the usage of the J kappa 1 gene in three proteins and the J kappa 2 gene in four of them. Position 96 contributed by the recombination of the V kappa and J kappa gene segments showed the presence of four different amino acid residues. Both anti-PSL2 and anti-PSL3 bind efficiently to all separated L chains when analyzed by the Western blot technique, and the binding was inhibited specifically by the corresponding peptides. The results reveal that the majority of human IgM-RF light chains are derived from a single germ line V kappa gene or a family of closely related V kappa III germ line genes, and express two "primary structure-dependent" CRI, which are largely dependent on the amino acid sequence of the second and third light chain CDR.     

Complete heavy and light chain variable region sequence of anti-arsonate monoclonal antibodies from BALB/c and A/J mice sharing the 36-60 idiotype are highly homologous

Structural and serologic studies on murine A/J monoclonal anti-arsonate antibodies resulted in the identification of a second idiotype family (Id36-60) in addition to the predominant idiotype family (IdCR). Id36-60, unlike IdCR, is a dominant idiotype in the BALB/c strain but is a "minor" idiotype in the A/J strain. The complete heavy and light chain variable region (VH and VL) amino acid sequences of a representative Id36-60 hybridoma protein from both the A/J and BALB/c strains have been determined. There are only four amino acid sequence differences between the VH of antibody 36-60 (A/J) and antibody 1210.7 (BALB/c). Two of these differences arise from single nucleotide changes in which the A/J and BALB/c Id36-60 VH germline gene sequences differ. The two other differences are the result of somatic mutation in hybridoma protein 36-60. In addition, Id36-60 heavy chains employ the same D and JH3 segments in both strains. The entire Vk2 VL of 36-60 and 1210.7 differ by only two amino acids, suggesting that like the heavy chains, they are derived from highly homologous VL genes. The same Jk segment is used in both antibodies. A comparison of the amino acid sequence data from Id36-60-bearing hybridomas suggests that a heavy chain amino acid difference accounts for the diminished arsonate binding by the 1210.7 hybridoma protein. Because the 1210.7 heavy chain is the unmutated product of the BALB/c VH gene, somatic mutation in VH may be required to enhance Ars affinity in this system.     

The association of various D elements with a single-immunoglobulin VH gene segment: influence on the expression of a major cross-reactive idiotype

A large fraction of the anti-p-azophenylarsonate antibodies of strain A/J mice share a major cross-reactive idiotype (IdCR). Structural analysis of monoclonal antibodies expressing this idiotype (IdCR+) indicates that a particular combination of variable region gene segments (Vk, Jk, VH, D, and JH) encodes the variable regions of the light and heavy chains of these IdCR+ antibodies. With the use of serologic methods, hybridoma cell lines have been isolated that produce monoclonal antibodies lacking IdCR determinants (IdCR-), but that are derived from most of the same combination of variable region gene segments that encode IdCR+ monoclonal antibodies. Structural analysis of these IdCR- monoclonal antibodies demonstrates that they are very homologous to each other and to IdCR+ monoclonal antibodies with respect to VH and VL sequences, but are markedly different from IdCR+ monoclonal antibodies in their utilization of D region segments. Comparisons of antigen avidity of these IdCR+ and IdCR- antibodies indicates that conservation of D region structure is not crucial for effective antigen binding. These results indicate the importance of the D region in idiotypy in the IdCR system and demonstrate the variation permitted in D region structure while maintaining antigen recognition.     

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.     

Expression of three cross-reactive idiotypes on rheumatoid factor autoantibodies from patients with autoimmune diseases and seropositive adults

Approximately one-half of human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors (RF] from unrelated individuals with cryoglobulinemia coordinately express three cross-reactive idiotypic antigens (CRI). The CRI are detected with: 1) monoclonal antibody 17.109, which recognizes a conformation-dependent CRI on K-light chains; and 2) two rabbit anti-peptide antibodies that react with primary sequence-dependent CRI (PSL2 and PSL3) corresponding to the conserved second and third K-chain complementarity-determining regions, respectively. In the present experiments, the structural features of polyclonal RF autoantibodies from diverse patients with rheumatoid arthritis and from those with primary Sj?gren's syndrome, and from seropositive elderly subjects without overt autoimmune diseases, were investigated with these three defined anti-CRI reagents. The pattern of expression of the CRI differed among patient groups. Only the RF autoantibodies from Sj?gren's syndrome patients frequently displayed all three CRI. However, the RF from nearly every subject tested, including patients with rheumatoid arthritis, were enriched in the primary sequence-dependent PSL2-CRI as compared to RF-depleted Ig from the same subjects. Amino acid sequence analysis of monoclonal IgM-RF indicates that PSL2-CRI-positive light chains probably represent the products of a single Vk gene. Therefore, a proportion of the polyclonal RF from different autoimmune states may represent somatic variants of this germ-line RF Vk gene which retain the PSL2 sequence as a common element.     

Structural studies on induced antibodies with defined idiotypic specificities. VII. The complete amino acid sequence of the heavy chain variable region of anti-p-azophenylarsenate antibodies from A/J mice bearing a cross-reactive idiotype.

This paper reports the complete amino acid sequence of the variable region of heavy chains derived from A/J anti-p-azophenylarsenate antibodies bearing a cross-reactive idiotype. The structure of this induced idiotypically defined antibody is homogeneous and provides the first direct evidence that heritable idiotypes are defined chemical entities. There are certain similarities between the structure of this murine antibody and the human myeloma protein Eu as well as guinea pig anti-p-azophenylarsonate antibodies. The amino acid sequence of approximately 15% of the constant region of this IgG1 molecule is also described. Combined with our studies of the variable region of the light chains of these molecules, this study represents the first complete V domain structure of an induced idiotypically defined antibody with heritable characteristics.     

Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q

Enterococcus faecium L50 grown at 16 to 32 degrees C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47 degrees C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Havarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47 degrees C and maximally detected at 47 and 37 to 47 degrees C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25 degrees C and are not detected at 37 degrees C or above.     

The rheumatoid factor reactivity of a human IgG monoclonal autoantibody is encoded by a variant V kappa II L chain gene

To determine the genetic and molecular basis for rheumatoid factor (RF) autoantibody reactivity in patients with destructive, erosive arthritis, we established a human lymphoblastoid cell line (hRF-1) from a patient with polyarthritis that produced an IgG RF mAb, mAb hRF-1. Studies of isolated H and L chains showed that the specificity of RF reactivity is conferred by mAb hRF-1 L chains. The L chain gene was cloned from a cDNA library prepared from hRF-1 cells. The nucleotide sequence was similar to known V kappa II L chains except for a two nucleotide change corresponding to a change of two amino acids in an invariable region of FR3. A germ-line gene with one of the nucleotide changes was identified by polymerase chain reaction in multiple cell lines, including K562 that does not rearrange Ig genes, but the other nucleotide change appeared to be due to mutation. Either or both of these amino acid changes may contribute to the RF reactivity, because an antibody with the same V kappa II L chain except for these two amino acid changes in FR3 did not have RF reactivity. The RF reactivity of isolated L chains from mAb hRF-1 was confirmed by transfecting COS cells with an expression vector encoding the hRF-1 kappa-chain and showing that the secreted k-chains had RF reactivity. Expression of this variant V kappa II L chain gene may form the basis for RF autoantibody reactivity in some patients.     

Nucleotide sequence of the B1-hordein gene of barley Hordeum vulgare L

The gene encoding B1 hordein of Hordeum vulgare (cv. Donetsky 4) was cloned and entirely sequenced. It contains no introns and codes for of 293 amino acids long polypeptide with molecular weight 33418. Our clone differs from the previously sequenced B1 hordein genes in some positions within the coding region (there are 4 nucleotide changes and a 12 bp deletion, as compared with the pB11 cDNA clone, and 5 nucleotide changes, as compared with the pBHP184 genomic clone). These changes result in polymorphism of amino acid sequences at 5 positions. 5'-flanking region contains putative regulatory and promoter sequences and differs from that of the pBHP184 clone in 3 positions.     

The majority of human monoclonal IgM rheumatoid factors express a "primary structure-dependent" cross-reactive idiotype

Genetic studies of human immunoglobulin variable regions have been hampered by the lack of anti-idiotypic antibodies that recognize specific heavy and light chain variable region sequences. Sixty percent of human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors [RF]) from unrelated individuals share a cross-reactive idiotype (CRI) termed Wa. In previous experiments in which we used an enzyme-linked immunosorbent assay, we reported that a synthetic peptide (PSL2), corresponding to the second hypervariable region in the kappa light chain of a monoclonal IgM-RF (Sie), induced rabbit antibodies reactive with several RF paraproteins. In the present experiments, to avoid interference due to the human IgM-RF binding toward rabbit IgG, the reactivity of the anti-PSL2 antibody to the separated heavy and light chains of multiple IgM proteins and Bence-Jones proteins was assessed by the Western blot technique. The PSL2-induced anti-CRI reacted well with the separated kappa chains from 10 out of 12 IgM-RF, zero out of four light chains from IgM proteins lacking anti-IgG activity, and one out of six kappa Bence-Jones proteins. The results show that the PSL2-CRI is associated with RF and is not a kappa subgroup marker. Furthermore, a comparison of the reported light chain sequences of the PSL2-CRI-positive IgM-RF suggests that the majority of human IgM-RF light chains derive from a single germ-line VK gene or from a family of closely related VK genes that is highly conserved in the human population. Synthetic peptide-induced anti-CRI provide a potent tool for analyzing the genetic basis of CRI and abnormal autoantibody production in humans.