Comparative analysis of human and mouse immunoglobulin variable heavy regions from IMGT/LIGM-DB with IMGT/HighV-QUEST

Abstract

Background: Immunoglobulin (IG) complementarity determining region (CDR) includes VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3. Of these, VH CDR3 plays a dominant role in recognizing and binding antigens. Three major mechanisms are involved in the formation of the VH repertoire: germline gene rearrangement, junctional diversity and somatic hypermutation. Features of the generation mechanisms of VH repertoire in humans and mice share similarities while VH CDR3 amino acid (AA) composition differs. Previous studies have mainly focused on germline gene rearrangement and the composition and structure of the CDR3 AA in humans and mice. However the number of AA changes due to somatic hypermutation and analysis of the junctional mechanism have been ignored.

Methods: Here we analyzed 9,340 human and 6,657 murine unique productive sequences of immunoglobulin (IG) variable heavy (VH) domains derived from IMGT/LIGM-DB database to understand how VH CDR3 AA compositions significantly differed between human and mouse. These sequences were identified and analyzed by IMGT/HighV-QUEST (http://www.imgt.org), including gene usage, number of AA changes due to somatic hypermutation, AA length distribution of VH CDR3, AA composition, and junctional diversity.

Results: Analyses of human and murine IG repertoires showed significant differences. A higher number of AA changes due to somatic hypermutation and more abundant N-region addition were found in human compared to mouse, which might be an important factor leading to differences in VH CDR3 amino acid composition.

Conclusions: These findings are a benchmark for understanding VH repertoires and can be used to characterize the VH repertoire during immune responses. The study will allow standardized comparison for high throughput results obtained by IMGT/HighV-QUEST, the reference portal for NGS repertoire.

Figure 1

Number of amino acid (AA) changes of all human (n = 9340) and mouse (n = 6657) sequences is shown. A Total number of AA changes in the V regions of VH domain. B The number of AA changes observed in FR1, CDR1, FR2, CDR2, and FR3 of VH domain, with the mean ± SD. The p values were determined using ANOVA. All statistically significant differences are indicated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 2

Average values of AA composition calculated for all human (n = 9340) and mouse (n = 6657) VH CDR3 sequences in percent shows the difference from overall amino acid composition in human and murine. The amino acids are arranged by relative hydrophobicity values as found in Kyte-Doolittle scale [35]. The overall amino acid composition differed significantly between human and murine VH CDR3 ( χ² test, 19 degrees of freedom, p < 0.001). Comparing the frequencies of individual amino acid residues, the definite level of significance was better than *0.05%, **0.001%, or ***0.00001% using χ² test and post-hoc analysis as described by Collis et al. [36].

Figure 3

The frequency of individual amino acids at the specific ten positions for all human (n = 9340) and mouse (n = 6657) VH CDR3 sequences is shown. The color menu for amino acids is according to IMGT [37]. CDR3 positions are shown according to the IMGT unique numbering. A human. B mouse.

Figure 4

The V-D-J junctional diversity created by P region, exonuclease trimming and N region addition significantly contributes to a large VH CDR3 repertoire. A The numbers of N, P nucleotides added at the V-D-J junctions and nucleotides deleted by exonuclease trimming are shown, with the mean ± SD. The p values were determined using ANOVA. All statistically significant differences are indicated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. B The numbers of N1, N2, P3'V, P5'D and P5'J nucleotides added at the V-D-J junctions and nucleotides deleted at the 3'V, 5'D, 3'D and 5'J by exonuclease trimming are shown, with the mean ± SD. The p values were determined using ANOVA. All statistically significant differences are indicated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.