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

Abstract

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 B3GLCT gene encoding for a β3-glucosyltransferase that catalyzes the attachment of glucose via a β1-3 glycosidic linkage to O-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 B3GLCT gene, b3glcta and b3glctb. The b3glcta and b3glctb 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. In vitro glucosylation assays demonstrated that extracts from wildtype embryos contain active b3glct enzyme capable of transferring glucose from UDP-glucose to an O-fucosylated TSR, indicating functional conservation with human B3GLCT. To determine the developmental role of the zebrafish genes, single and double b3glct knockouts were generated using TALEN-induced genome editing. Extracts from double homozygous b3glct-/- embryos demonstrated complete loss of in vitro b3glct activity. Surprisingly, b3glct-/- homozygous fish developed normally. Transcriptome analyses of head and trunk tissues of b3glct-/- 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 B3GLCT/b3glct genes is conserved in vertebrates. At the same time, complete b3glct deficiency in zebrafish appears to be inconsequential and possibly compensated for by a yet unknown mechanism.

Fig 1. Exonic structure, genomic context and multiple species alignment of B3GLCT/b3glct.

(A) The two zebrafish orthologs of B3GLCT show overall similar exonic arrangement. The number of each exon is located within each box and the size of the exon (in base pairs) is shown above each exon. The 5’ and 3’ UTRs are indicated preceding the first ATG and following the stop codon (TAA/TAG). White indicates the N-terminal signal sequence, light grey indicates the stem region and dark grey indicates the catalytic domain. The vertical black bar in exon 12 of each gene indicates the location of nucleotides encoding for the catalytic tri-aspartic acid residues. Horizontal lines underneath the zebrafish genes indicate previously annotated sequence and sequence identified in this study. (B) Schematic of genomic context for B3GLCT/b3glct. (C) Multiple species alignment of B3GLCT orthologs from human (NP_919299), mouse (NP_001074673), Xenopus (NP_001072551), and zebrafish. Blue bar indicates signal peptide, green indicates stem region and orange indicates catalytic core. Grey shading of amino acids indicates conservation. The DxD motif is boxed in red.

Fig 2. Embryonic expression of zebrafish b3glct genes.

(A) RT-PCR analysis of b3glct expression demonstrates robust expression of both b3glcta (left panel) and b3glctb (middle panel) at different stages of development in whole embryos as well as various embryonic tissues at 48-hpf (right panel). Controls included pitx2c as negative control for 0-hpf, rhodopsin as negative control for the lens, beta-actin as positive control for all tissues and H₂O as negative contamination control for all reactions. (B) In-situ hybridization analysis of b3glcta and b3glctb expression demonstrates broad expression in 24-120-hpf embryos with enrichment in the developing eyes, fins, brain, craniofacial region and somites. aer–apical ectodermal ridge, ase–anterior segment of the eye, b–brain, cmz–ciliary marginal zone, crc–craniofacial cartilage, e–eye, f–fins, h–heart, le–lens, sm–skeletal muscles.

Fig 3. Genetic disruption of b3glct.

(A) Schematic of b3glct genes indicating TALEN target sites (exon 1 and 12 for b3glcta and exon 12 for b3glctb, black arrows). The predicted protein product resulting from TALEN mediated disruption is shown. Editing events in the first exon of b3glcta are predicted to disrupt nearly the entire coding region of the transcript. For both b3glcta and b3glctb, editing in the 12^th exon is predicted to result in loss of most of the catalytic domain including the catalytic core and KDEL-like ER retention signal. Blue (SP)- Signal Peptide, Green (SR)- Stem Region, Orange (CD)- Catalytic Domain. (B) Images of zebrafish embryos at 5-dpf and adult zebrafish showing no gross morphological defects associated with loss of b3glct. (C) Functional evaluation of wild-type and mutant b3glct by in vitro β3-glucosyltransferase assays. Left panel- the endogenous β3-glucosyltransferase activity toward O-fucosylated TSR3 is dependent on the amount of protein in the wild type zebrafish homogenate; control reaction with 10 μg homogenate was performed using unmodified TSR3. Right panel- the endogenous β3-glucosyltransferase activity toward O-fucosylated TSR3 in the homogenate of double homozygous b3glct embryos is profoundly reduced compared with that in the wild type zebrafish homogenate; control reactions were performed using unmodified TSR3. Assays were performed in triplicate. Error bars indicate s.d.