Evolution of surrogate light chain in tetrapods and the relationship between lengths of CDR H3 and VpreB tails

Abstract

In the mammalian immune system, the surrogate light chain (SLC) shapes the antibody repertoire during B cell development by serving as a checkpoint for production of functional heavy chains (HC). Structural studies indicate that tail regions of VpreB contact and cover the third complementarity-determining region of the HC (CDR H3). However, some species, particularly bovines, have CDR H3 regions that may not be compatible with this HC-SLC interaction model. With immense structural and genetic diversity in antibody repertoires across species, we evaluated the genetic origins and sequence features of surrogate light chain components. We examined tetrapod genomes for evidence of conserved gene synteny to determine the evolutionary origin of VpreB1, VpreB2, and IGLL1, as well as VpreB3 and pre-T cell receptor alpha (PTCRA) genes. We found the genes for the SLC components (VpreB1, VpreB2, and IGLL1) only in eutherian mammals. However, genes for PTCRA occurred in all amniote groups and genes for VpreB3 occurred in all tetrapod groups, and these genes were highly conserved. Additionally, we found evidence of a new VpreB gene in non-mammalian tetrapods that is similar to the VpreB2 gene of eutherian mammals, suggesting VpreB2 may have appeared earlier in tetrapod evolution and may be a precursor to traditional VpreB2 genes in higher vertebrates. Among eutherian mammals, sequence conservation between VpreB1 and VpreB2 was low for all groups except rabbits and rodents, where VpreB2 was nearly identical to VpreB1 and did not share conserved synteny with VpreB2 of other species. VpreB2 of rabbits and rodents likely represents a duplicated variant of VpreB1 and is distinct from the VpreB2 of other mammals. Thus, rabbits and rodents have two variants of VpreB1 (VpreB1-1 and VpreB1-2) but no VpreB2. Sequence analysis of VpreB tail regions indicated differences in sequence content, charge, and length; where repertoire data was available, we observed a significant relationship between VpreB2 tail length and maximum DH length. We posit that SLC components co-evolved with immunoglobulin HC to accommodate the repertoire - particularly CDR H3 length and structure, and perhaps highly unusual HC (like ultralong HC of cattle) may bypass this developmental checkpoint altogether.

Keywords: Lambda5; VpreB; evolution; pre-B cell receptor; pre-T cell receptor alpha; surrogate light chain.

Figure 1

The human SLC may not accommodate an ultralong CDR H3. (A) A human fragment antigen-binding region (F_ab; PDB 5ZMJ; doi: 10.2210/pdb5ZMJ/pdb) is shown next to a human pre-BCR composed of an immunoglobulin heavy chain (HC) paired with the surrogate light chain (SLC; PDB 2H3N; doi: 10.2210/pdb2H3N/pdb). (B) For comparison, a cow ultralong F_ab (PDB 5IJV; doi: 10.2210/pdb5IJV/pdb) is shown next to ultralong HC of that Fab with the human SLC from (A). The structures are colored as follows: purple = HC, red = CDR H3, light green = light chain, dark green = VpreB1, blue = λ5, yellow = SLC residues that are important for interacting with the HC, dotted black line = part of VpreB1 tail that was truncated to obtain the crystal structure. Note that the VpreB1 tail may not engage the apex of the CDR H3 knob. (C) Amino acid alignment of VpreB1 (top), VpreB2 (middle), and IGLL1 tail regions, isoelectric point (pI), and amino acid tail length from eutherian mammal species. Though mice have two variants (v1 and v2) of VpreB1, neither mouse nor human have VpreB2. Note the high pI (more basic) tail of cattle VpreB1 and low pI (more acidic) tail of cattle VpreB2 compared to other species. Residues are shaded based on similarity using a Blosum62 scoring matrix (threshold = 1, gaps ignored; highlights indicate similarity: black = 100% similar; dark gray = 80 – 100% similar; light gray = 60 – 80% similar; white = <60% similar).

Figure 2

Model of surrogate light chain evolution in tetrapods. Cladogram of major tetrapod vertebrate groups illustrating the evolution of VpreB3, pre-T cell receptor alpha (PTCRA) chain, and genes used to construct surrogate light chain (VpreB1, VpreB2, and IGLL1). Multiple species inform each branch (see Supplementary Tables 1 - 3 for accession numbers, amino acid sequences, and genomic locations of genes for included species). Colored rectangles indicate gene emergence for the gene named within the box. Similarly colored lines represent gene loss or gene absence (e.g., solid line at Mammalia; due either to gene loss or genome fragmentation) within that branch. Tree topology is based on mammalian phylogeny reported in Murphy et al. (34); individual branches that expand on this published tree are informed by published phylogenies of that group (–38). Evolutionary distances are not to scale. Asterisks (*) next to a branch name indicates a species for which we examined genomes for synteny. Branch name colors coordinate groups within the cladogram to those of sequence alignments and phylogenies (see Supplementary Figures 1 - 7 ). Created with BioRender.com

Figure 3

Locus synteny of VpreB3 and surrogate light chain (VpreB1, VpreB2, IGLL1) genes in tetrapod vertebrates. (A) Putative orthologs of both VpreB (A: sid1, light purple) and IGLL1 (light orange, B: si:ch211-1a19.2) in the zebrafish (Danio rerio) genome (B) In tetrapod vertebrates, relevant genes are shown in pink (VpreB3), purple (VpreB1), green (VpreB2), and orange (IGLL1). Polygons containing syntenic genes found in two or more species contain a number corresponding to the gene name. Polygons containing genes for uncharacterized proteins are empty. A solid line represents contiguous genomic sequence (chromosome or unplaced scaffold) and is denoted by the chromosome (Chr) or scaffold (Scf) to which it belongs. A line ending in a diamond shape indicates the end of a chromosome or scaffold and no further genes occur in that direction. Orientation of chromosomes or scaffolds is indicated by the 5’ or 3’ labels at the ends of the solid lines, and gene polygons point in their transcriptional direction. We indicate blocks of syntenic genes with the number of genes in a box below the line and long, unaligned regions with the genomic distance between syntenic genes. Colored lines above the polygons denote syntenic gene blocks, and lines containing an arrow indicate the syntenic block is inverted compared to caecilian. A red line below the polygons indicates an insertion of non-syntenic genes. The location of the lambda light chain locus (IGL, if present) is indicated by a gray rectangle. Orthologs between species are aligned vertically, but distances are not to scale. Polygons containing variable segments of lambda (LV) or kappa (KV) light chains and those containing lambda constant regions (LL) are reported with the annotated V or C segment number. In hedgehog, “Om” refers to the annotated gene omega, an alternate gene card name for VpreB2, that do not encode the VpreB2 protein. All other gene names are denoted by a number within the polygon (Polygon #: Gene name 1: MIF; 2: SLC2A11; 3: DERL3; 4: SMARCB1; 5: MMP11; 6: CHCH210; 7: C22orf15; 8: ZNF70; 9: RGL4; 10: PDCH15; 11: CA15L; 12: TOP3B; 13: BABAM1; 14: OAS1; 15: PRAME; 16: ZNF280A; 17: ZNF280B; 18: SGLT1; 19: SLC5A4; 20: SLC5A1; 21: FDG4; 22: SLC25A1; 23: CLTCL1; 24: VPS29L; 25: WSCD2; 26: C12orf43; 27: RPL4; 28: PRAME12L; 29: PRAME6L; 30: ESS2; 31: GSC2; 32: SPAG61; 33: DGCR6; 34: ZNF596L; 35: ZNF596; 36: CDK20; 37: NUTM2D; 38: PPM1F; 39: MAPK1; 40: YPEL1; 41: HIRA; 42: PPIL2; 43: SPSPON; 44: THADA; 45: DGCR2; 46: DDX51; 47: SLC2A5; 48: SLC2A9; 49: TSSK2; 50: GALNT9; 51: DDT; 52: FAM200BIL; 53: WRD18; 54: ARRDC2; see Supplementary Table 3B for gene names). Created with BioRender.com

Figure 4

Locus synteny of VpreB3 and surrogate light chain (VpreB1, VpreB2, IGLL1) genes in cattle and their closest relatives. Genes are shown in pink (VpreB3), purple (VpreB1), green (VpreB2), and orange (IGLL1). Polygons containing syntenic genes found in two or more species contain a number corresponding to the gene name (see Figure 3 for genes represented by each numbered polygon). Polygons containing genes for uncharacterized proteins are empty. A solid line represents contiguous genomic sequence (chromosome or unplaced scaffold) and is denoted by the chromosome (Chr) or scaffold (Scf) to which it belongs. A line ending in a diamond shape indicates the end of a chromosome or scaffold and no further genes occur in that direction. Blocks of syntenic genes are indicated with the number of genes in a box below the line and long, unaligned regions with the genomic distance between syntenic genes. Blocks of syntenic genes are indicated with the number of genes in a box below the line. Orientation of chromosomes or scaffolds is indicated by the 5’ or 3’ labels at the ends of the solid lines, and gene polygons point in their transcriptional direction. Colored lines above the polygons denote syntenic gene blocks, and lines containing an arrow indicate the syntenic block is inverted compared to horse. The location of the lambda light chain locus (IGL, if present) is indicated by a gray rectangle. An arrow between genomes of two species indicates a gene that is translocated in one species. Orthologs between species are aligned vertically, but distances are not to scale. See Figure 1 and Supplementary Table 3B for a list gene names. A model cladogram is provided for reference. Tree topology is informed by published phylogenies (38). Created with BioRender.com

Figure 5

Locus synteny of VpreB3 and surrogate light chain (VpreB1, VpreB2, IGLL1) genes in Glires (rabbits and rodents) and their closest relatives. Genes are shown in pink (VpreB3), purple (VpreB1), green (VpreB2), and orange (IGLL1). Polygons containing syntenic genes found in two or more species contain a number corresponding to the gene name (see Figure 3 for gene names represented by each numbered polygon). Polygons containing genes for uncharacterized proteins are empty. A solid line represents contiguous genomic sequence (chromosome or unplaced scaffold) and is denoted by the chromosome (Chr) or scaffold (Scf) to which it belongs. A line ending in a diamond shape indicates the end of a chromosome or scaffold and no further genes occur in that direction. Orientation of chromosomes or scaffolds is indicated by the 5’ or 3’ labels at the ends of the solid lines, and gene polygons point in their transcriptional direction. Colored lines above the polygons denote syntenic gene blocks, and lines containing an arrow indicate the syntenic block is inverted compared to dog. A red line below the polygons indicates an insertion of non-syntenic genes that includes this second VpreB gene in Glires. The location of the lambda light chain locus (IGL, if present) is indicated by a gray rectangle. Orthologs between species are aligned vertically, but distances are not to scale. A model cladogram is provided for reference. Tree topology is informed by published phylogenies (–38). Created with BioRender.com

Figure 6

VpreB2 tails are longer in species with elongated DH gene segments. (A) VpreB2 tail lengths are highly correlated with maximum DH length (r= 0.81; p=0.0499; n = 6). (B) However, this relationship disappears when cattle are removed from the dataset (r=-0.029; p=0.957; n = 5).

Figure 7

VpreB tail length is negatively correlated with isoelectric point (pI) of tail residues. (A) pI and length of VpreB1 tail regions show a weak negative correlation (r = -0.31; p = 0.021; n = 56), though (B) this relationship is not due to pI of VpreB1 tail residues (r = -0.22; p = 0.09; n = 56). (C) pI and length of VpreB2 tail regions show a moderate negative correlation (r = -0.59; p = 0.026; n = 24), and this (D) relationship was unrelated to pI of VpreB2 tail residues (r = -0.25; p = 0.24; n =24).

Figure 8

Isoelectric point (pI) of IGLL1 tails increases with increasing CDR H3 length. (A) pI of IGLL1 tails was highly correlated with maximum CDR H3 length (r= 0.73; p=0.01; n = 11). (B) This relationship remains even when cattle (with ultralong DH segments) are removed from the dataset (r=-0.78; p=0.0072; n = 10).