Molecular evolution and in vitro characterization of Botryllus histocompatibility factor

• Published in: Immunogenetics (New York) Vol. 67; no. 10; pp. 605 - 623
• Main Authors: Taketa, Daryl A., Nydam, Marie L., Langenbacher, Adam D., Rodriguez, Delany, Sanders, Erin, De Tomaso, Anthony W.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-10-2015; Springer Nature B.V
• Subjects: Alleles; Allergology; Amino Acid Sequence; Animals; Biomedical and Life Sciences; Biomedicine; Blotting, Western; Botryllus schlosseri; Cell Biology; Developmental biology; Evolution, Molecular; Gene Expression Profiling; Gene Function; Genetic Variation; Haplotypes; HEK293 Cells; Human Genetics; Humans; Hypotheses; Immunology; In Situ Hybridization, Fluorescence; Localization; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Major Histocompatibility Complex - genetics; Microscopy, Confocal; Molecular Sequence Data; Original Paper; Protein Isoforms - genetics; Protein Isoforms - metabolism; Proteins; Red Fluorescent Protein; Urochordata - genetics; Urochordata - immunology; United States > US; Allorecognition; histocompatibility factor (BHF); Cellular localization; Selection; Botryllus schlosseri; Botryllus histocompatibility factor (BHF)
• ISSN: 0093-7711; 1432-1211
• DOI: 10.1007/s00251-015-0870-1

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.