Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase <i>B3GLCT</i> gene mutated in Peters Plus Syndrome

Abstract

<div><p>Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, short stature, brachydactyly, characteristic facial features, developmental delay and other highly variable systemic defects. Classic PPS is caused by loss-of-function mutations in the <i>B3GLCT</i> gene encoding for a β3-glucosyltransferase that catalyzes the attachment of glucose via a β1–3 glycosidic linkage to <i>O</i>-linked fucose on thrombospondin type 1 repeats (TSRs). B3GLCT was shown to participate in a non-canonical ER quality control mechanism; however, the exact molecular processes affected in PPS are not well understood. Here we report the identification and characterization of two zebrafish orthologs of the human <i>B3GLCT</i> gene, <i>b3glcta</i> and <i>b3glctb</i>. The <i>b3glcta</i> and <i>b3glctb</i> genes encode for 496-aa and 493-aa proteins with 65% and 57% identity to human B3GLCT, respectively. Expression studies demonstrate that both orthologs are widely expressed with strong presence in embryonic tissues affected in PPS. <i>In vitro</i> glucosylation assays demonstrated that extracts from wildtype embryos contain active b3glct enzyme capable of transferring glucose from UDP-glucose to an <i>O</i>-fucosylated TSR, indicating functional conservation with human B3GLCT. To determine the developmental role of the zebrafish genes, single and double <i>b3glct</i> knockouts were generated using TALEN-induced genome editing. Extracts from double homozygous <i>b3glct</i>^<i>-/-</i> embryos demonstrated complete loss of <i>in vitro</i> b3glct activity. Surprisingly, <i>b3glct</i>^<i>-/-</i> homozygous fish developed normally. Transcriptome analyses of head and trunk tissues of <i>b3glct</i>^<i>-/-</i> 24-hpf embryos identified 483 shared differentially regulated transcripts that may be involved in compensation for b3glct function in these embryos. The presented data show that both sequence and function of <i>B3GLCT/b3glct</i> genes is conserved in vertebrates. At the same time, complete <i>b3glct</i> deficiency in zebrafish appears to be inconsequential and possibly compensated for by a yet unknown mechanism.</p></div