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The Journal of Immunology, 2003, 171: 3886-3894.
Copyright © 2003 by The American Association of Immunologists

Molecular Signatures of Anti-nuclear Antibodies: Contributions of Specific Light Chain Residues and a Novel New Zealand Black V{kappa}1 Germline Gene 1

Zhiyan Liang, Cui Chen and Chandra Mohan2

Simmons Arthritis Research Center and Center for Immunology, University of Texas Southwestern Medical School, Dallas, TX 75235


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the Ig H chains of anti-nuclear Abs (ANA) have been described to possess certain shared molecular signatures, it remains unclear whether the L chains of these Abs also possess distinctive molecular features. The present study examines this by generating and analyzing two comprehensive murine Ig L chain databases, one consisting of 264 monoclonal ANAs and the other consisting of 145 non-ANAs, drawn from previously published work. Importantly, clonal replicates were represented only once each, so as to minimize bias. ANAs and non-ANAs did not differ in V{kappa} family or J{kappa} gene usage, nor in their mutation frequencies. Interestingly, the L chains of ANAs exhibited differential usage of certain complementarity-determining region residues, arising almost entirely from the increased usage of certain V{kappa} germline genes, notably, V{kappa} ai4 among anti-dsDNA ANAs, V{kappa}23–45 among anti-ssDNA ANAs, and V{kappa}21–12 among non-ANAs. Finally, prompted by the increased prevalence of a particular V{kappa}1 family sequence among ANAs, we proceeded to clone a novel New Zealand Black V{kappa}1 germline gene, named bb1.1, which appears to be frequently used to encoded anti-ssDNA Abs. Collectively, these studies underline the potential contribution of particular V{kappa} germline genes in promoting or thwarting DNA binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Anti-nuclear Abs (ANAs)3 play an essential role in the pathogenesis of systemic lupus erythematosus (1, 2, 3, 4). Although we have learned a great deal about the pathogenic potential and clinical significance of ANAs, there are still several outstanding questions concerning ANAs that remain unanswered. Importantly, the structural features of ANAs that set them apart from other Abs, and confer DNA philicity and pathogenic potential remain unclear. Over the past two decades, several investigators have rescued and characterized mAbs with different nuclear Ag fine specificities from several different lupus-prone murine strains (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39), as previously reviewed (40, 41, 42). Recently, the H chains (HC) of these different mAbs have been consolidated into a common database for sequence analysis (43). This HC database consists of nearly 300 well-defined murine mAbs, including anti-ssDNA, anti-dsDNA, and anti-nucleosome ANAs. Computational analysis using this sequence database has been useful in revealing the molecular signatures that may be peculiar to the HC of ANA. Thus, compared with the HC of non-ANA control Abs (drawn from the Kabat database), ANA HCs demonstrated several significant differences in their complementarity-determining regions (CDR), particularly in the incidence of charged or polar residues. In addition, the HC of anti-dsDNA ANAs differed significantly from those of anti-ssDNA ANAs in having: 1) more D residues at H31 and more Y residues at H33, in their CDR1 regions; 2) significantly different distribution of polar residues at the CDR2 positions, H53, H55, and H56; and 3) more R residues in the CDR3 positions, H95–H100.

Although the HC may play a dominant role in conferring DNA reactivity, it is clear that L chains (LC) have the potential to modulate this in an important way (44, 45, 46, 47, 48, 49, 50, 51, 52). In transfection experiments in which the HC of an anti-DNA Ig transgene (Tg) (3H9) was kept constant, it was observed that only a minor fraction of V{kappa} genes had the potential to veto DNA binding (47). Extending these studies further, Radic and colleagues have cataloged the different LC that can confer ssDNA, dsDNA, or cardiolipin reactivity when paired with the 3H9 HC (48). More recently, in anti-DNA HC-only Tg models, Weigert and colleagues (49) have made the intriguing observation that anionic LC genes (such as V{kappa}9, V{kappa}12/13, V{kappa}20, V{kappa}21d, and V{kappa}38c) had the capacity to veto DNA binding, in the context of different DNA-philic HC. In particular, aspartates in the CDR1 regions of these V{kappa} germline genes were implicated as playing an important role in this phenomenon. The notion that specific LC residues can serve to contact DNA directly is further supported by site-directed mutagenesis studies. For instance, other investigators (52, 53) have demonstrated that mutating away particular D residues in LC CDR1 can augment DNA binding. Although the roles of different LCs and specific LC residues have been elegantly demonstrated by the above transfection, transgenic, and mutagenesis studies, the potential role that any such differences might play in spontaneously arising ANAs remains unclear.

Although spontaneously arising ANAs have been rescued as mAbs by previous investigators, earlier studies aimed at discerning any potential molecular fingerprints that may be peculiar to ANAs have been constrained by a couple of limitations. First, most studies have focused on limited sets of ANAs, often including clonally related members. Second, they have invariably used the entire Kabat collection of Abs (54) as their control database. However, the Kabat database harbors a significant fraction of multimember clonal trees, large collections of anti-hapten Abs, and a substantial content of anti-nuclear Abs, all of which could have potentially skewed any derived results. Finally, in the absence of sequence information from clonal trees (in which members may differ in residue usage at particular positions), it has traditionally been difficult to compute precise values for the somatic mutation frequencies, as the near-complete V{kappa} germline repertoire had not been elucidated until recently (55, 56, 57, 58, 59).

The present study was designed to address the above caveats in a couple of different ways. First, we have constructed a composite ANA LC database, drawing from the published literature of murine monoclonal ANAs; this database consists of 139 anti-ssDNA, 103 anti-dsDNA, and 22 anti-nucleosome ANAs. Second, a normal or control database has also been constructed, consisting of 145 non-ANA LCs extracted from the National Center for Biotechnology Information (NCBI)/GenBank database, in which each Ag specificity was represented only once. Third, this study takes advantage of the recently elucidated germline V{kappa} repertoire, to re-evaluate the potential contribution of germline-encoded vs mutated residues, in the composition of anti-nuclear Abs. By comparing these two Ab databases, this study aims to determine whether the LC used to encode spontaneously arising ANAs differ from those encoding non-ANAs, with respect to the frequencies of V{kappa} and J{kappa} gene use, somatic mutation frequencies, and the amino acid usage patterns, particularly in the CDR regions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Criteria for inclusion in the control, non-ANA LC database

A convenient choice for a normal or non-ANA LC database is that compiled by Kabat (54). Given the limitations of using the Kabat database, as discussed above, we constructed an abridged version of the normal database, using the following criteria:

The Ag specificities of the Abs deposited in the NCBI/GenBank database were systematically screened. For each target Ag, only one mAb was selected for inclusion in the control, non-ANA database. Thus, for example, although there were hundreds of anti-nuclear protein Abs deposited in GenBank, only one anti-nuclear protein Ab (i.e., the first electronic hit during the search process) was included in the control, non-ANA database. Searching through the entire NCBI database, we identified a total of 145 mAbs with specificities targeted to different (nonnuclear) Ags.

Abs with documented reactivity to DNA or histones, or other nuclear Ags (e.g., Sm) were omitted from the non-ANA database.

Abs with unknown antigenic specificities were not included in the non-ANA database.

Sequences that were incomplete in their CDR regions were also excluded.

This regime reduced the NCBI/GenBank collection of Abs into a much smaller non-ANA database, consisting of 145 LC sequences, with nonoverlapping antigenic specificities.

Criteria for inclusion in the ANA LC database

A total of 35 published reports of spontaneously arising murine ANA mAbs was reviewed (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39). LC sequences of all Abs that fulfilled the criteria listed below were selected for inclusion in the ANA database:

Abs that exhibited definite reactivity to ssDNA, with documented absence of reactivity to dsDNA, were included in the database, and classified as anti-ssDNA ANAs.

Abs that exhibited reactivity with mammalian dsDNA, independent of whether or not they also reacted with ssDNA or nucleosomes, were included in the database, and classified as anti-dsDNA ANAs. Indeed, most anti-dsDNA Abs also exhibited reactivity with ssDNA and nucleosomes as well, where tested.

Abs that reacted preferentially with histone/DNA or nucleosomal complexes, rather than with histone-free dsDNA or DNA-free histones, were included in the database, and classified as anti-nucleosome ANAs.

Multimember clonal trees were represented only once in the ANA database. Approximately 80% of the anti-ssDNA mAbs, 67% of the anti-dsDNA Abs, and 74% of the anti-nucleosome Abs in this database represented singleton clones. For each of the remaining multimember families, a single representative was picked at random for inclusion in the database, without any reference to how strongly mutated it was (relative to the other clonal relatives), so as to avoid any systematic bias in representing these multimember clonal families.

Importantly, the following sequences were omitted from the ANA database:

Abs that were loosely classified as being chromatin reactive or ANA + ve, or anti-DNA, without any further analysis of their fine specificity using defined nuclear Ag substrates.

Abs that were described as anti-nucleosome without any demonstration of a lack of reactivity to histone-free dsDNA or DNA-free histones.

Abs that exhibited a weak or "±" grade of reactivity to nuclear Ags, as described in the original communications.

ANAs that had incomplete CDR sequence information.

Using the above criteria, a total of 139 anti-ssDNA, 103 anti-dsDNA, and 22 anti-nucleosome ANAs was identified for inclusion in the ANA database.

Features cataloged in the databases

For each Ab in the ANA and non-ANA database, the following information was cataloged: literature reference; strain/model of origin; antigenic specificity; GenBank accession number, if available; V{kappa} family usage (see below); J{kappa} usage; and the CDR1, CDR2, and CDR3 sequences. Each Ig LC sequence was also blasted against the publicly accessible IgBlast database of mouse Ig sequences at National Institutes of Health/NCBI (http://www.ncbi.nlm.nih.gov/igblast/), to determine the closest germline V{kappa} gene of origin. Comparing these sequences with the recently published repertoire of germline V{kappa} genes allowed us to identify residues that were somatically mutated. The CDR position and numbering scheme adopted matched that used by the NCBI IgBlast database. Of particular note, most of the entries in the original manuscript (and in the Kabat database) had used an older numbering nomenclature, and had to be realigned so as to match the ANA database, as well as the IgBlast reference database. Both the non-ANA and ANA LC databases are freely available to all interested investigators from C. Mohan, or from our website (http://www3.utsouthwestern.edu/mohan/).

Cloning of novel bb1 genes

VK1 bb1-related germline genes were isolated by PCR amplification of tail DNA from New Zealand Black (NZB), New Zealand White (NZW), and C57BL/6 (B6) mice using a 5' framework region 1 sense primer (5'-GACCCAAACTCCACTCTCC-3'), and a 3' recombination signal sequence antisense primer (5'-TGTTAGGGTCTGTATCACTGTG-3'). Amplified products were gel purified and cloned using the TA cloning system (Invitrogen, Carlsbad, CA). Ten individual colonies from each strain were purified for sequence analysis. The newly identified V{kappa}1 bb1-like germline gene of NZB origin (assigned the name bb1.1) has been deposited into GenBank (accession number pending).

Data analysis

In addition to comparing all ANAs with non-ANAs, anti-ssDNA Abs and anti-dsDNA Abs were also compared with each other, with respect to several different parameters. There were too few anti-nucleosome Abs available for a careful analysis of the molecular signatures pertaining to this group of ANAs. The frequencies of V{kappa} and J{kappa} gene usage, as well as the frequencies of individual amino acid residues at the different CDR1, CDR2, and CDR3 positions were compared between the different groups of Abs, using 2 x 2 {chi}2 tests, with one degree of freedom. Where applicable, the Fisher’s exact test was applied. For the analysis of residue differences at the different CDR positions, we adopted a more stringent criterion of p < 0.01 for determining statistical significance, because we were performing multiple comparisons of the usage frequencies of the most dominant residues at each CDR position. All statistical comparisons were performed using SigmaStat (Jandel Scientific, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A total of 264 previously documented, spontaneously arising murine ANAs (comprised of 139 anti-ssDNA, 103 anti-dsDNA, and 22 anti-nucleosome Abs) was compared with 145 non-ANAs (drawn from the NCBI/GenBank database), with respect to their LC V{kappa} and J{kappa} usage. As depicted in Fig. 1A, a wide spectrum of LC V{kappa} genes was used to encode ANAs and non-ANAs. In both Ab groups, V{kappa}1 genes constituted the largest group, accounting for about one-fifth to one-quarter of all Abs, with V{kappa}4/5 genes representing a close second. Following the V{kappa}1 and V{kappa}4/5 families, V{kappa}8, V{kappa}19/28, and V{kappa}21 genes represented the next three most frequently used families, each accounting for ~10% of all Abs (Fig. 1A). All other V{kappa} families accounted for <5% each, in all Ab groups. The only difference of significance in V{kappa} family usage was seen in the V{kappa}4/5 family; these V{kappa} genes were used significantly more frequently to encode anti-dsDNA Abs (25% of all Abs), compared with the corresponding frequencies among non-ANAs (18.5% of all Abs, p < 0.04), or anti-ssDNA Abs (12.6% of all Abs, p < 0.03).



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  		FIGURE 1. V{kappa} and J{kappa} gene usage in murine ANAs and non-ANAs. As detailed in Materials and Methods, a nonredundant panel of non-ANAs (n = 145) and ANAs (n = 264) was assembled, drawing from 35 previously published manuscripts (5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 ). The ANAs included 139 clearly well-defined anti-ssDNA Abs and 103 anti-dsDNA Abs. These different panels were compared for their respective usage frequencies of V{kappa} gene family (A), V{kappa} germline genes (B), and J{kappa} genes (C). Statistical comparisons between groups were performed using the Student’s t test (*, indicates p < 0.05; ***, indicates p < 0.001).

 
Recently, the genomic V{kappa} locus/repertoire (of the B6-derived, Ig{kappa} c allotype) has been elucidated, almost completely (55, 56, 57, 58, 59). This germline repertoire, amounting to a total of 93 V{kappa} genes (73 of which are clearly expressed, while the remaining 20 are potentially functional), is readily accessible through the NCBI IgBlast database. This gave us the opportunity to ascertain whether ANAs differed from non-ANAs in the specific germline genes they used. Overall, we found that all groups of Abs used different germline genes to a fairly similar extent. Thus, in all Ab groups, the V{kappa}1 germline gene, bb1, was the most frequently used, accounting for ~60–80% of all V{kappa}1-encoded Abs. A similar usage of germline genes was also seen in the other V{kappa} families, with three exceptions. As depicted in Fig. 1B, the V{kappa}4 germline gene, ai4, was significantly more frequent among anti-dsDNA Abs (9.5% of all Abs) than among anti-ssDNA ANAs (0%, p < 0.001), or among non-ANAs (3.4%, p < 0.07). Second, anti-ssDNA ANAs were encoded more frequently by the V{kappa}23–45 germline gene than were anti-dsDNA ANAs (p < 0.05). Finally, the V{kappa}21-12 germline gene was more common among non-ANAs (p < 0.05) than among anti-ssDNA and anti-dsDNA Abs (Fig. 1B).

All groups of Abs demonstrated a similar distribution of J{kappa} usage, with J{kappa}1 and J{kappa}2 predominating, with each accounting for about one-third of all Abs in each database (Fig. 1C). Although no differences in J{kappa} usage were noted, it was interesting to observe that the combined usage of J{kappa}1 and J{kappa}2 was approximately twice that of J{kappa}4 and J{kappa}5 combined, in both Ab databases, independent of V{kappa} gene usage. This observation supports the notion that the J{kappa}1 and J{kappa}2 genes may be preferentially rearranged (in ANAs and non-ANAs alike), compared with the more distal J{kappa} genes, consistent with previous observations (60). In addition, inasmuch as the utilization frequency of 3' J{kappa} genes is an indicator of the extent of LC editing (61), receptor editing does not seem to have played a differential role in the genesis of ANAs vs non-ANAs.

The positioning of certain polar residues at specific CDR positions has been shown to be particularly important for specificity in ANA HCs (40, 41, 42, 43). We wondered whether similar residue differences might also distinguish ANA LCs. In contrast to the previous findings with ANA HCs, LCs of ANAs and non-ANAs used similar residues at almost all CDR positions. Table I lists the few exceptions that exceeded the cutoff for statistical significance (p < 0.01, corrected for multiple comparisons). Most of these differences involved polar residues, or residues capable of engaging in H-bond formation. Both the differences of significance in CDR1 involved serine residues. Thus, at the CDR1 position, L27d, S was far less frequent among anti-ssDNA ANAs, compared with the other Ab groups, including non-ANAs (p < 0.04). At the next CDR1 position, L27e, S was far more frequent among anti-dsDNA ANAs (85%) than among anti-ssDNA ANAs (68%, p < 0.013) and among non-ANAs (61%, p < 0.003).


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  		Table I. Summary of significant residue differences at LC CDR positions of ANAs and non-ANAs

 
The residues used to encode the CDR2 region were fairly similar in all groups of Abs, with one exception. Whereas Y was rarely used at L53, it was present in 17% of all anti-dsDNA ANA LCs, this frequency being significantly higher than that in all other Ab groups. Likewise, most of the CDR3 residues were similar between the different groups of Abs, with two exceptions. T at L92 and Y at L94 were approximately twice as common among anti-ssDNA ANAs and anti-dsDNA ANAs, respectively, compared with the frequencies among non-ANAs (Table I). In addition to examining the usage of individual residues at the different CDR positions, we also ascertained the respective frequencies of different classes of residues (grouped based on physical similarities, e.g., R/K, D/E, N/R/Y, etc.) at each position in the different groups of Abs. However, using this comparison, no additional differences of significance (at p < 0.01) were noted between the different Ab groups.

Given that the LCs of ANA exhibited significantly altered usage of certain residues at a few selected CDR positions, we next examined whether these differences were germline encoded, or were the products of potential somatic mutation. The recent elucidation of the near-complete murine V{kappa} germline repertoire allows one to ascertain the germline V{kappa} counterpart for most Ab sequences, with a high degree of certainty. All sequences in the non-ANA and ANA databases were compared with the publicly available repository of germline V{kappa} sequences (http://www.ncbi.nlm.nih.gov/igblast/), to ascertain the potential contribution of somatic mutation. Whereas the CDR1 and CDR2 regions exhibited mutation frequencies of ~6–8% (at the amino acid level), the CDR3 regions exhibited nongermline sequence variations at frequencies of ~12% (Table II). Interestingly, anti-dsDNA ANAs tended to have fewer mutations in their CDR1 regions compared with the non-ANAs (p < 0.002). Any potential differences in the rate of silent mutations were not assessed.


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  		Table II. Amino acid mutation frequencies in ANA and non-ANA LC sequences

 
Although the overall mutation frequencies may not have been very different; it was conceivable, however, that Ag-driven selection of mutated residues may have been important in shaping residue differences at selected CDR positions. When we focused on the few CDR residue differences exhibited by the ANAs (as listed in Table I), we found that almost all of the differences highlighted in Table I were germline encoded, with minimal contribution from somatic mutation (Fig. 2). Because most of the CDR residues that were differentially used between the ANAs and the non-ANAs (Table I) were germline encoded, it follows that ANAs and non-ANAs may differ in the usage of LC germline genes that differ at these particular CDR positions. In this context, we have already noted earlier (Fig. 1B) that the usage frequencies of three V{kappa} germline genes differed significantly between ANAs and non-ANAs Therefore, we next examined whether the differential usage of these three (or perhaps other) germline genes may have contributed to the CDR residue differences depicted in Table I.



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  		FIGURE 2. Germline vs somatic origin of LC CDR residues that distinguish ANAs from non-ANAs. ANAs differed from non-ANAs in their residue usage frequencies at five different LC CDR positions, as detailed in Table I. Plotted are the fraction of non-ANAs, anti-ssDNA ANAs, and anti-dsDNA ANAs that possessed the indicated amino acids at the respective CDR positions, as a consequence of mutation (filled bar), or as germline-encoded residues (open bar). The absolute numbers of Abs represented in each bar can be deduced from Table I.

 
With respect to the CDR1 position, L27d, it was interesting to observe that anti-ssDNA ANAs were encoded more frequently by V{kappa}23-45 (which does not bear an S residue at L27d) and less frequently by V{kappa} ai4 (which bears an S at L27d), as shown in Fig. 1B. In addition, the germline genes V{kappa}12-44 and ba9 (both of which bear S at L27d) were used more frequently among non-ANAs than among anti-ssDNA Abs, although these differences did not reach statistical significance. Together, these differences in germline V{kappa} usage accounted for the significantly reduced frequencies of S at L27d, among anti-ssDNA ANAs. We next examined the possible origins of the residue differences seen at the CDR1 position, L27e. It is clear from Fig. 1A that V{kappa}4/5 genes were more frequently used among anti-dsDNA Abs. Of relevance, most of these V{kappa}4/5 germline genes, including ai4, possessed an S residue at L27e. Indeed, if one were to exclude all sequences encoded by the ai4 germline gene from all databases, the residue usage difference at this CDR1 position was no longer apparent (data not shown).

The increased frequency of Y at the CDR2 position, L53, among anti-dsDNA Abs was not accounted for by the increased frequency of ai4 germline genes. However, because all Y residues at L53 were germline encoded (Fig. 2), we proceeded to search for germline genes that were endowed with a Y residue at L53. In fact, only two germline genes in the entire V{kappa} repertoire were so endowed: V{kappa}19-15 and V{kappa}19-17. Indeed, both these germline genes were more frequently used to encode anti-dsDNA ANAs, compared with the other Abs, although these differences fell short of statistical significance. Hence, this difference in germline gene usage accounted for the increased frequency of Y at L53 among the anti-dsDNA Abs.

Next, we examined the potential origin of the observed residue usage differences in the CDR3 regions of ANA LCs. Compared with the other groups of Abs, anti-ssDNA Abs were more frequently encoded by V{kappa}1 and V{kappa}2 genes, although these differences did not reach statistical significance (Fig. 1A). However, these are the only two V{kappa} gene families that bear T at the CDR3 position, L92. Indeed, the increased usage of the V{kappa}1 germline gene, bb1, and the V{kappa}2 germline gene, bd2, totally accounted for the elevated frequency of T residues at L92 among anti-ssDNA Abs. Likewise, as is evident from Fig. 1B, anti-dsDNA ANAs were more frequently encoded by V{kappa}4/5 (most of which possessed Y at the CDR3 position, L94), and much less frequently by V{kappa}21-12 and 23-45 (both of which lack Y at L94). Collectively, these germline gene usage differences accounted for the significant residue usage differences observed at L94 as well.

Thus, all five of the residue usage differences that apparently distinguished the LC CDRs of ANAs from those of non-ANAs were accounted for by differential usage of particular V{kappa} germline genes, predominantly, with negligible contribution from somatic mutation. Next, we asked whether any of the other LC CDR positions exhibited differential usage of particular amino acid residues, as a direct consequence of somatic mutation. In so doing, two additional findings of interest were uncovered. First, there was an increased frequency of mutations to N, R, or Y residues at positions L24, L27c, L27d, L34, and L50 among the ANAs, compared with the non-ANAs. However, these differences did not reach statistical significance. Second, in all groups of Abs, particularly among the ANAs, we noted a couple of clusters of independently derived Abs sharing three or more apparently mutated (i.e., nongermline-encoded) amino acid residues at the same respective positions. We reasoned that these were likely to represent novel germline genes, not represented in the NCBI IgBlast database.

Table III illustrates the most prominent of such clusters, comprised of Abs using the V{kappa}1 germline gene, bb1, which represents the most frequently used germline gene in all Ab groups. Listed are all non-ANAs (n = 18) and anti-ssDNA Abs (n = 30) that were assigned by NCBI Igblast as being derived from the V{kappa}1 bb1 germline gene. Table III illustrates several interesting points. First, certain positions, notably L27e, appeared to be mutational hot spots in both groups of Abs. Second, mutations to N/R residues appeared to be more frequent at this position among the ANAs than among the non-ANAs, although this difference did not reach statistical significance. Finally, several bb1-related LC sequences encoding anti-ssDNA Abs (all of NZB/NZW origin) shared Y at L34, R at L50, F at L89, and G at L91, residues that were clearly not germline encoded by the bb1 gene. Indeed, five of these LCs were identical in sequence, despite originating from different mice (and from independent studies).


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  		Table III. Non-ANA and anti-ssDNA ANA sequences that were assigned as being derived from the bb1 Vk germline genea

 
The odds that the same four somatic mutations might have arisen in 10 independent Ab isolates by random chance alone are infinitely small (less than 1 in 1025). It appeared far more likely that this represented a novel bb1-related germline gene of NZB/NZW origin. In contrast to its prevalence among anti-ssDNA ANAs, this novel, putative germline gene was not apparent among the non-ANAs, and was fairly uncommon among anti-dsDNA ANAs. Indeed, when one reviews the germline gene usage of anti-ssDNA ANAs, this novel bb1-related gene turns out to be the second most frequently used germline gene, next only to the usage frequency of the bona fide bb1 gene, as is apparent from Table III. Given its prominence in encoding (NZB/NZW-derived) anti-ssDNA Abs, we next proceeded to clone the germline counterpart of this novel gene.

Because NZW and B6 mice share the same Ig{kappa} allotype (62), and because these particular sequences were not seen among the Kabat-derived non-ANAs (that were mostly of BALB/c or B6 origin), we hypothesized that the NZB strain may harbor this putative, novel bb1-like germline gene. Table IV lists the bb1-like PCR products cloned and sequenced from NZB genomic DNA. As we had hypothesized, it is clear that the NZB strain harbors a novel V{kappa}1 germline gene that differs from the bb1 gene in the NCBI IgBlast database, at the 4 particular aa residues indicated (first two entries in Table IV). We have accorded the name, bb1.1, to this new V{kappa}1germline gene; importantly, because the NZB repertoire does not appear to possess the canonical bb1 gene, it appears that this novel germline gene, bb1.1, may be the only bb1-like gene that NZB mice might possess. This approach also rescued several isolates bearing bl1, another V{kappa}1 germline gene that is closely related to the bb1 gene in sequence (i.e., the last four entries in Table IV). Finally, a couple of additional genes that were closely related to the bb1.1 and b11 germline genes were also isolated in this analysis (Table IV). Importantly, the same approach when applied to NZW and B6 genomic DNA yielded the original or canonical bb1 sequence, but showed no evidence of the novel bb1.1 gene (data not shown).


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  		Table IV. Sequences of bb1-related germline genes amplified from NZB tail DNAa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Over the past two decades, about three dozen independent studies of various murine models of lupus have resulted in the generation of several hundred monoclonal ANAs. Collectively, these ANAs constitute a wonderful resource that could potentially shed light on the molecular structures of different fine specificities of ANAs. This is indeed important to elucidate because these different ANA fine specificities may have very different pathogenic roles in orchestrating disease. Although sequence/specificity comparisons and correlations of a limited nature have been reported in the past (40, 41, 42), a collective analysis of all ANA LCs studied to date and a comprehensive database of these sequences do not exist. The present study strives to achieve this goal. We have generated a relatively unbiased database of ANA LCs, by collecting together the LC sequences of all published ANAs with clearly defined specificities and complete sequences, and by eliminating all clonal siblings.

There are certainly pros and cons in adopting such an approach. Of note, because the mAbs investigated in this study had originated from nine different mouse strains, molecular features that were peculiar to selected Ig V{kappa} haplotypes, or strain backgrounds, may have been obscured by this pooling of data. Indeed, the sequences in this ANA database were encoded by V{kappa} genes from at least three different haplotypes (62): Ig{kappa} a (MRL), Ig{kappa} b (NZB), and Ig{kappa} c (C57BL/6, BALB/c, NZW, BXSB, SWR, C3H strains). Thus, whereas most of the ANAs were derived from the MRL/lpr and BWF1 strains, most of the non-ANAs (in the Kabat database) were derived from the normal B6 and BALB/c strains. Although pooling of all the available ANA LC information might potentially uncover molecular signatures of anti-ssDNA and anti-dsDNA ANAs that are quite independent of strain-to-strain (and mouse-to-mouse) variations, and potential HC contributions, it would clearly be critical to derive and compare large panels of ANAs and non-ANAs from the same strains of mice in the future.

The present study shows that the V{kappa} repertoire is fairly similar between ANAs and non-ANAs. In addition, the same V{kappa} germline genes, with a few interesting exceptions, were used to encode both types of Abs. The tendency to overuse V{kappa}4 ai4, notably among anti-dsDNA Abs, is particularly interesting. The only residue that distinguishes ai4 from all other V{kappa} germline genes is the presence of R at L93; it is presently not clear whether this single difference qualifies this germline gene as an avid DNA binder. Likewise, the significant underutilization of V{kappa}21-12 among the ANAs is also very intriguing given the observation that the relatively anionic V{kappa}21 gene has been demonstrated to be an excellent editor that can veto DNA binding in anti-DNA HC Tg models (49). Finally, ANAs and non-ANAs also revealed very few residue usage differences in their CDR regions; importantly, almost all of these differences were germline encoded.

It is particularly interesting and important to compare the findings of this study with those of previous reports. The CDR1 regions of ANA LCs have been described to exhibit several important characteristics. Radic and Weigert (41) have suggested that the CDR1 lengths of anti-DNA Abs may be about 2 aa residues longer than those of non-ANAs. In the present study, ANAs and non-ANAs had similar CDR1 lengths (13.48 vs 13.40, p > 0.05). In addition, comparative studies of limited sets of ANAs with the Kabat collection of Abs as well as computer-modeling studies had suggested that polar or charged residues in the amino-terminal half of LC CDR1 (positions 27f to 32) may be important in facilitating DNA binding (5, 38, 48, 62, 63, 64). These include R at L27f, N/R at L28 and L29, N/R/K at L30, N/R/K/S at L31, and N/R/Y at L32; interestingly, D/E at these positions have been suggested to influence dsDNA and nucleosome binding differentially (38, 48, 62).

Before comparing the above reports with the findings presented in this study, it should be pointed out that most of the above studies have used a CDR1 position-numbering nomenclature that is slightly different from the one adopted by the IgBlast database as well as by the present study. In particular, whereas the middle two to three residues in the CDR1 regions were right justified (to positions L29-L31) in the older nomenclature, they are left justified (to positions L27c-L27e) in the newer nomenclature. Thus, whereas the respective frequencies at positions L24-L27b and L32-L34 are directly comparable, care should be exercised when comparing the frequencies elsewhere in CDR1. In the present study, besides the altered frequencies of S at L27d and L27e (which have already been discussed above; Table I), three additional trends that echoed the findings of previous reports were of interest in the CDR1 regions of ANAs:

At L30, anti-dsDNA ANAs exhibited increased frequencies of K (33.3%) and Y (17.6%), compared with the non-ANAs (23 and 9%, respectively).

At L31, anti-dsDNA ANAs exhibited increased frequencies of N (28.3%), apparently at the expense of S residues (17%), compared with the non-ANAs (16 and 26%, respectively).

At L32, anti-dsDNA ANAs exhibited increased frequencies of N (9.8%), compared with the non-ANAs (5%).

Although these differences in polar residues resonated well with the findings noted in some of the earlier reports, all fell short of reaching statistical significance. Clearly, larger numbers of Abs would be required to confirm the significance of these suggestive trends.

Fewer reports have implicated LC CDR2 residues as being important in ANAs. However, polar residues have been suggested as being potentially important, particularly at L50 and L52–56 (63). With the exception of Y at L53 (Table I), the only other trend of note was the marginal elevation of R (14%) and S (18%) residues at L50, among anti-dsDNA ANAs, compared with the non-ANAs (which exhibited usage frequencies of 10 and 11%, respectively); however, this difference fell short of statistical significance. The LC CDR3 domain has also been implicated by previous investigators as being potentially important in mediating DNA binding. In particular, comparative studies of limited sets of ANAs with the Kabat collection of Abs, as well as computer-modeling studies, had suggested that polar or charged residues at L92-L96 may be particularly critical (5, 38, 41, 48, 63, 64). This included D/N at L92, E at L93, Y at L94, Q at L95, and R/Y at L96. However, besides the changes reported in Table I, no further residue usage differences were noted in the CDR3 regions of ANA LCs. For instance, previous studies have suggested that R residues at L96 arising as a consequence of V{kappa}-J{kappa}1 joining were particularly prominent among anti-dsDNA Abs (8, 40, 41). However, in the present analysis, the frequency of R at L96 was almost identical between anti-dsDNA Abs and the non-ANA controls, arising in both cases as a consequence of rearrangement to J{kappa}1.

Most of the earlier studies cited above were based on comparing limited sets of ANAs (often including entire clonal trees) against the entire Kabat collection of Abs, or on modeling studies based on a single Ab. Thus, it is clearly possible that several of the reported ANA-specific residue differences may have been applicable to limited sets of Abs only, perhaps in the context of specific HC partners. For example, although the V{kappa} usage and residue differences reported by Radic and colleagues (41, 47, 48) may be operational in the context of the 3H9 HC partner, this may not be true in the context of other HC partners.

Weigert and colleagues (49) have suggested that the average isoelectric point of the entire combining surface of the LC may be an important determinant of DNA reactivity, particularly in the context of different HC partners. Thus, for example, whereas the anionic LC genes have varying degrees of potential to veto DNA binding, cationic LC genes, such as V{kappa}1 and V{kappa}4, may have the potential to facilitate DNA binding. However, in our analysis, the anti-ssDNA Abs, anti-dsDNA Abs, as well as the total ANA database of Abs did not differ significantly from the non-ANA Abs in their mean pI values across any of the CDR regions (data not shown). Thus, it appears that the global charge of the LC CDR regions may not be an independent determinant of DNA philicity, but may constitute a property that may potentially become important in the context of particular HC partners.

It is acknowledged that the present study design may not have been optimal in uncovering the true extent of contribution from somatic mutation, as multimember clones were represented by only one sequence each. It is perhaps not a surprise that almost all of the observed sequence differences between ANAs and non-ANAs were germline in origin. Even if the decision to represent clonally expanded families with one member each might have underestimated the potential contribution of somatic mutation, the fact that ~75% of the ANAs described in this work were singleton clones indicates that a large fraction of the spontaneously arising ANA clones in murine lupus possesses germline-encoded molecular differences that distinguish them from non-ANAs. Superimposed on these germline-encoded differences, somatic mutation is likely to imprint a further set of molecular signatures on ANAs, as they become clonally expanded and undergo affinity maturation. The specific mutations that may confer selective advantage are likely to vary from clone to clone, as richly documented in the original reports (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39).

Although the BALB/c, C57BL/6, and the lupus-prone or lupus-facilitating strains, NZW, SWR, and BXSB, all bear the Ig{kappa}c haplotype (62), the MRL/lpr (Ig{kappa}a) and NZB (Ig{kappa}b) strains clearly bear different alleles at this locus. Thus, when studying ANAs from these different strains, it is important to consider the possibility that novel germline genes may account, in part at least, for some of the observed molecular differences. The description of the novel bb1-like gene of NZB origin, bb1.1, in this manuscript illustrates this. Among the four residues that distinguish bb1.1 from the canonical bb1 germline gene, Y at L34 and R at L50 are particularly interesting, because polar residues at these positions have been suggested by previous investigators as being more prominent among ANAs (5, 38, 48, 63, 64). Hence, it is tempting to posit that these molecular features of the NZB bb1.1 LC gene might have conferred increased DNA philicity to B cell receptors bearing them. From a broader perspective, the preferential use of certain germline genes to encode anti-ssDNA ANAs (e.g., V{kappa} bb1.1 or 23-45), anti-dsDNA ANAs (e.g., V{kappa} ai4), or non-ANAs (e.g., V{kappa}21-12) underlines the potential importance of germline-encoded residues in facilitating or vetoing DNA binding. Clearly, these predictions need to be verified using expression and mutagenesis studies.


   Acknowledgments
 
We thank Drs. William Garrard and Martin Weigert for critical reading of the manuscript and helpful discussions.


   Footnotes
 
1 Work in our laboratory is funded by grants from the National Institutes of Health (AR44894 and AI47460) and the National Arthritis Foundation. C.M. is a recipient of the Robert Wood Johnson Jr. Arthritis Investigator Award. Back

2 Address correspondence and reprint requests to Dr. Chandra Mohan, Simmons Arthritis Research Center, Department of Internal Medicine/Rheumatology, University of Texas Southwestern Medical Center, Mail Code 8884, Y8.204, 5323 Harry Hines Boulevard, Dallas, TX 75390-8884. E-mail address: Chandra.mohan{at}utsouthwestern.edu Back

3 Abbreviations used in this paper: ANA, anti-nuclear Ab; CDR, complementarity-determining region; HC, H chain; LC, L chain; Tg, transgene. Back

Received for publication November 22, 2002. Accepted for publication July 31, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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