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. 2015 Oct;67(10):605-23.
doi: 10.1007/s00251-015-0870-1. Epub 2015 Sep 11.

Molecular evolution and in vitro characterization of Botryllus histocompatibility factor

Daryl A Taketa  1 , Marie L Nydam  2 , Adam D Langenbacher  1 , Delany Rodriguez  1 , Erin Sanders  1   3 , Anthony W De Tomaso  4
Affiliations

Molecular evolution and in vitro characterization of Botryllus histocompatibility factor

Daryl A Taketa et al. Immunogenetics. 2015 Oct.

Abstract

Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.

Keywords: Allorecognition; Botryllus histocompatibility factor (BHF); Botryllus schlosseri; Cellular localization; Selection.

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Conflict of interest statement

Conflict of Interest

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Amino acid diversity of BHF. (a) DIVAA analysis (Rodi et al. 2004) of BHF sequences shows amino acid diversity of individuals in the Santa Barbara harbor based on 29 sequences. (b) Sequence of BHF (GenBank AGS14996.1) with the amino acid polymorphisms found in the Santa Barbara harbor population shown below. Bolded text represents significant changes in amino acid residues’ properties
Fig. 2
Fig. 2
mRNA-Seq expression profile. (a) Schematic representation of genomic structure (black) with overlapping EST hits (blue) with an E-value < 1e−10 and mRNA-Seq reads mapped to the genomic region visualized on IGV. Gray region in the mRNA-Seq represent conserved nucleotide while the colors represent the following nucleotides: red (T), blue (C), green (A), and orange (G). (b) RT-PCR of bhf isoform 2 on a 1% agarose gel with an expected size of 718 bp. (c–e) mRNA-Seq expression profile based on normalized counts across the blastogenic cycle (n = 4 per stage) for bhf isoform 1 (c), isoform 2 (d), cfuhcsec (e). (f) mRNA-Seq between ampullae tissue (n = 15) and whole colony (n = 28) for bhf isoform 1, isoform 2, and cfuhcsec. Error bars are standard error of normalized counts from DESeq with a false discovery rate = 10%
Fig. 3
Fig. 3
Expression localization via whole-mount FISH. (a–d) Confocal Z-stacks of adult colonies with either a bhf isoform 1 full length probe (a–c) or a bhf CΔ102 truncated probe that will bind both isoforms (d) on ampullae (a & d), resorbing zooid (blastogenic cycle stage D; b), and in the secondary bud (c). (e-g’’’) Confocal images on double labeled whole-mount FISH on juvenile colonies (<3 months; ~3–4 zooids) with probes designed against bhf CΔ102 and cfuhcsec. (e–e’’’) Confocal images of an ampullae. (f–f’’’) Z-stack of blood vessels. (g–g’’’) Z-stack of the endostyle. All samples were counterstained with DAPI (blue). White arrows indicate co-expressing cells of bhf CΔ102 and cfuhcsec. Dashed outlines mark the boundary of the indicated tissue. Amp = ampullae; RZ = resorbing zooid; E = endostyle; PB = primary bud; SB = secondary bud; V = blood vessel. Scale bar = 50 µm
Fig. 4
Fig. 4
Localization of BHF in HEK293T cells. (a–b) Confocal Z-stack image of mCherry:BHF (isoform 1) (a) or mCherry:BHF (isoform 2) (b). (c–d) Confocal slices of mCherry (c) or mCherry:BHF isoform 1 (d) expressing HEK293T cells (red) stained with a rabbit α-GPR50 antibody (green). All images have been counterstained with DAPI (blue). (e) Western blot of subcellular fractions (cytosol = Cyto; membrane = Mem) on transiently expressing mCherry (~29 kDa) or mCherry:BHF isoform 1 (~56 kDa) cells blotted with a rat α-mCherry, rabbit α-EGFR, or rabbit α-HSP90 antibody. The lower bands (~45 kDa) in the right panel are truncated mCherry:BHF from degradation or partially translated. Scale bar = 10 µm
Fig. 5
Fig. 5
BHF is localized to the inner side of the plasma membrane in cycloheximide treated HEK293T (a–d) and proteinase K protection assay (e). (a, a’) HEK293T negative control stained for endogenous GPR-50 (green) in the absence (a) or presence (a’) of Triton X-100. The epitope for GPR-50 is on the intracellular side of the plasma membrane. (b, b’) mCherry (red) transient expression. (c, c’) mCherry:BHF (isoform 1; red) transient expression. (d, d’) BHF:mCherry (isoform 1; red) transient expression. (b, c, d) Negative controls stained with a rat α-mCherry (green) and counterstained with DAPI (blue). (b’, c’, d’) Permeabilized with Triton X-100 then stained with a rat α-mCherry (green), mCherry (red), DAPI (blue). Scale bar = 10 µm. (e) Western blot from a proteinase K protection assay using a rat α-mCherry antibody to detect recombinant proteins in transient expressing cells
Fig. 6
Fig. 6
Confocal images of different truncation mutants of mCherry:BHF (a–f) or BHF:mCherry (f–j) in red. NΔ or CΔ represent which amino acid terminal end was truncated by x many amino acids. (a) Full length mCherry:BHF. (b) BHF CΔ52:mCherry. (c) BHF CΔ102:mCherry. (d) BHF CΔ152:mCherry. (e) BHF CΔ202:mCherry. (f) Full length BHF:mCherry. (g) NΔ50 BHF:mCherry. (h) NΔ100 BHF:mCherry. (i) NΔ150 BHF:mCherry. (j) NΔ200 BHF:mCherry. (k) Mutant construct of the 3 cysteines into serines within the first 50 aa of mCherry:BHF acquired with a 60× objective. (l) Same construct as (a) but repeated and acquired with a 60× objective. (m) Percentage of cells that showed nuclear localization in the mutant (n = 431 cells) and wildtype control (n = 478 cells). All images were counterstained with DAPI (blue). Scale bar = 10 µm
Fig. 7
Fig. 7
Nuclear localization of BHF. Confocal images of (a) mCherry:BHF (red) and stained with a mouse α-Lamin A/C antibody (green); (b) BHF:mCherry (red) with a mouse α-Lamin A/C stain (green); (c) BHF (isoform 2):mCherry; (d) BHF:FLAG detected with a mouse α-FLAG antibody (green). All images were counterstained with DAPI (blue). Scale bar = 10 µm
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