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Glucosyltransferase B3GLCT Gene Mutated in Peters Plus Syndrome

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Glucosyltransferase B3GLCT Gene Mutated in Peters Plus Syndrome RESEARCH ARTICLE Functional characterization of zebrafish orthologs of the human Beta 3- Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome Eric Weh1,2¤a, Hideyuki Takeuchi3¤b, Sanaa Muheisen1, Robert S. Haltiwanger3, Elena V. Semina1,2,4* 1 Department of Pediatrics and Children's Research Institute at the Medical College of Wisconsin, a1111111111 Milwaukee, Wisconsin, United States of America, 2 Department of Cell Biology, Neurobiology, and Anatomy at the Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America, 3 Complex a1111111111 Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America, a1111111111 4 Department of Ophthalmology at the Medical College of Wisconsin, Milwaukee, Wisconsin, United States a1111111111 of America a1111111111 ¤a Current address: Department of Ophthalmology and Visual Sciences, W.K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, United States of America ¤b Current address: Department of Molecular Biochemistry, Nagoya University School of Medicine, Nagoya, Japan. * [email protected] OPEN ACCESS Citation: Weh E, Takeuchi H, Muheisen S, Haltiwanger RS, Semina EV (2017) Functional Abstract characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular mutated in Peters Plus Syndrome. PLoS ONE 12 defects, short stature, brachydactyly, characteristic facial features, developmental delay (9): e0184903. https://doi.org/10.1371/journal. pone.0184903 and other highly variable systemic defects. Classic PPS is caused by loss-of-function muta- tions in the B3GLCT gene encoding for a 3-glucosyltransferase that catalyzes the attach- Editor: Christoph Englert, Leibniz Institute on aging β - Fritz Lipmann Institute (FLI), GERMANY ment 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 Received: May 25, 2017 mechanism; however, the exact molecular processes affected in PPS are not well under- Accepted: September 1, 2017 stood. Here we report the identification and characterization of two zebrafish orthologs of Published: September 19, 2017 the human B3GLCT gene, b3glcta and b3glctb. The b3glcta and b3glctb genes encode for Copyright: © 2017 Weh et al. This is an open 496-aa and 493-aa proteins with 65% and 57% identity to human B3GLCT, respectively. access article distributed under the terms of the Expression studies demonstrate that both orthologs are widely expressed with strong pres- Creative Commons Attribution License, which ence in embryonic tissues affected in PPS. In vitro glucosylation assays demonstrated that permits unrestricted use, distribution, and reproduction in any medium, provided the original extracts from wildtype embryos contain active b3glct enzyme capable of transferring glu- author and source are credited. cose from UDP-glucose to an O-fucosylated TSR, indicating functional conservation with Data Availability Statement: Microarray data are human B3GLCT. To determine the developmental role of the zebrafish genes, single and available from ArrayExpress (http://www.ebi.ac.uk/ double b3glct knockouts were generated using TALEN-induced genome editing. Extracts arrayexpress/) under accession number EMTAB- from double homozygous b3glct-/- embryos demonstrated complete loss of in vitro b3glct 5966. activity. Surprisingly, b3glct-/- homozygous fish developed normally. Transcriptome analy- Funding: This work was supported by the National ses of head and trunk tissues of b3glct-/- 24-hpf embryos identified 483 shared differentially Institutes of Health awards R01EY015518 (EVS), R01GM061126 (RSH), and P30EY001931 as well regulated transcripts that may be involved in compensation for b3glct function in these as funds provided by the Children's Hospital of embryos. The presented data show that both sequence and function of B3GLCT/b3glct Wisconsin (EVS). PLOS ONE | https://doi.org/10.1371/journal.pone.0184903 September 19, 2017 1 / 19 Zebrafish orthologs of B3GLCT Competing interests: The authors have declared genes is conserved in vertebrates. At the same time, complete b3glct deficiency in zebrafish that no competing interests exist. appears to be inconsequential and possibly compensated for by a yet unknown mechanism. Introduction Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, brachydactyly, short stature, characteristic facial features and developmental delay as well as other variable systemic defects affecting the skeletal, cardiovascular, genitourinary and central nervous systems [1±3]. The ocular defect most often associated with PPS is Peters anomaly (PA); however, other defects affecting the anterior segment of the eye have been reported [1±3]. PA is an adhesion between the cornea and the lens that results in a central cor- neal opacity, or leukoma, and can lead to blindness if not treated [4]. The causative gene for PPS was originally identified by Lesnik Oberstein in 2006 as B3GLCT (formerly B3GALTL) [5]. Since then, our and other groups have identified multiple B3GLCT mutations in patients with PPS and defined the phenotype associated with B3GLCT mutations [2, 5±15]. B3GLCT mutations result in classic PPS which is characterized by a triad of features including anterior segment defects (100%; Peters anomaly in 85%), short stature (100%), and brachydactyly (95%), as well as variable other highly penetrant features such as developmental delay (84%), congenital heart defects (40%), cleft lip/palate (37%) and other anomalies [3]. At the same time, PPS-like cases (often clinically diagnosed as PPS) which dem- onstrate overlapping phenotypes but lack one or more of the key triad of features seen in clas- sic PPS were found to not be caused by mutations in B3GLCT [2, 3, 16±18]. Human B3GLCT is located on chromosome 13q12.3 and is divided into 15 exons encoding for a 498 amino acid protein. B3GLCT mRNA is expressed in a variety of different adult human tissues including heart, brain, lung, kidney, and skeletal muscle [19]. Based on sequence similar- ity and amino acid conservation at the catalytic site the encoded protein is predicted to follow the stereotypical domain architecture of other members of the GT31 glycosyltransferase family. The gene was initially described as encoding for a β1-3-galactosyltransferase based on sequence similarity [19], however this was revised once the catalytic activity of the enzyme was deduced [20, 21]. These studies demonstrated that B3GLCT is a β3-glucosyltransferase that catalyzes the attachment of glucose to O-linked fucose via a β1±3 glycosidic linkage on thrombospondin type 1 repeats (TSRs). TSRs can be found on over 60 human proteins, however only 49 of them con- tain the required consensus sequence to be fucosylated and subsequently modified by B3GLCT [22]. Proteins that have been shown to be modified by B3GLCT include Thrombospondin-1 [23], Thrombospondin-2 [24], Properdin [25], F-spondin [26], ADAMTS-13 [27], ADAMTSL- 1 [28], and ADAMTS-5 [29]. Properdin has been shown to lack proper modification in patients with PPS [25] yet is still secreted, although at a somewhat lower amount than normal. Addition- ally, PPS patients do not display any features of Properdin deficiency, an X-linked immune dis- order [30]. These data suggest that glucosylation by B3GLCT may not be necessary for the secretion and/or function of all possible targets of this enzyme. B3GLCT was found to participate in a non-canonical ER quality control pathway by Vasu- devan and colleagues [31]. Their report found that modification by B3GLCT marks properly folded TSR motifs and is necessary for proper secretion of a subset of TSR containing proteins. Still, the underlying molecular mechanisms leading to PPS through disruption of this pathway are not understood and animal models of B3glct deficiency are yet to be developed. In this manuscript we report the characterization of two zebrafish orthologs of the human B3GLCT PLOS ONE | https://doi.org/10.1371/journal.pone.0184903 September 19, 2017 2 / 19 Zebrafish orthologs of B3GLCT gene: b3glcta and b3glctb. We show that both zebrafish orthologs are well conserved with the human B3GLCT gene/protein and strongly expressed throughout the developing zebrafish embryo. We also demonstrate a lack of a visible morphological phenotype in b3glcta-/-, b3glctb-/- or b3glcta-/-; b3glctb-/- genetic lines generated via TALEN-mediated genome editing. Further studies of a potential compensatory mechanism via transcriptome analyses identified a variety of differentially regulated genes which may be capable of compensating for the lack of b3glct during zebrafish development. Results Identification and characterization of the zebrafish b3glcta and b3glctb A search for zebrafish orthologs of the human B3GLCT gene (NP_919299.3) using NCBI BLASTp and zebrafish databases identified two different protein-coding sequences. The first putative zebrafish ortholog (b3glcta, XM_001339763.3) comprised a 1240-bp tran- script predicted to encode for a protein of 300 amino acids in length (XP_001339799) and was located on chromosome 15. The second putative zebrafish ortholog (b3glctb, XM_687 506.5) included a 1686-bp mRNA sequence predicted to encode for a protein of 452 amino acids in length (XP_692598.5) and was located on chromosome 10 (Fig 1). When the iden- tified b3glcta and b3glctb sequences were aligned
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