The promiscuous binding pocket of SLC35A1 ensures redundant transport of CDP-ribitol to the Golgi
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100789
ISSN1083-351X
AutoresBenoît Ury, Sven Potelle, Francesco Caligiore, Matthew R. Whorton, Guido T. Bommer,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency. The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency. The dystrophin-associated glycoprotein 1 (DAG1) gene gives rise to a single protein that is subsequently cleaved into two fragments that remain attached to each other. The C-terminal part, β-dystroglycan, is a transmembrane protein anchored to the actin cytoskeleton through interaction with the dystrophin protein (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1158) Google Scholar). The N-terminal extracellular part, α-dystroglycan, binds to extracellular matrix components, like laminin, agrin, perlecan, or neurexin (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1158) Google Scholar). This interaction depends on the assembly of a complex glycan on α-dystroglycan. Defects in several enzymes required for the biogenesis of this glycan lead to a group of congenital syndromes characterized by muscle, brain, and eye symptoms. These syndromes have been coined dystroglycanopathies and present a vast clinical spectrum ranging from mild muscular weakness with late onset to severe congenital muscular dystrophy with brain and eye involvement (muscle–eye–brain disease, Walker–Warburg syndrome, and Fukuyama congenital muscle dystrophy) (2Endo T. Glycobiology of alpha-dystroglycan and muscular dystrophy.J. Biochem. 2015; 157: 1-12Crossref PubMed Scopus (88) Google Scholar, 3Live D. Wells L. Boons G.J. Dissecting the molecular basis of the role of the O-mannosylation pathway in disease: Alpha-dystroglycan and forms of muscular dystrophy.Chembiochem. 2013; 14: 2392-2402Crossref PubMed Scopus (19) Google Scholar, 4Michele D.E. Barresi R. Kanagawa M. Saito F. Cohn R.D. Satz J.S. Dollar J. Nishino I. Kelley R.I. Somer H. Straub V. Mathews K.D. Moore S.A. Campbell K.P. Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies.Nature. 2002; 418: 417-422Crossref PubMed Scopus (677) Google Scholar, 5Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (109) Google Scholar). The investigation of genes mutated in affected patients has led to the discovery of many steps in this process and a better understanding of α-dystroglycan glycosylation as well as more generally of protein O-mannosylation. The α-dystroglycan protein contains three domains: the C-terminal domain, which binds to the extracellular domain of β-dystroglycan, the mucin domain characterized by several mucin-type-O-glycosylation sites as well as a key O-mannosyl glycan important for ligand binding (referred to later as core M3), and the N-terminal domain that is required for complete glycosylation and is subsequently shed by proteolytic cleavage (5Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (109) Google Scholar). The ligand-binding epitope of α-dystroglycan is assembled on the core M3 glycan, which has been extensively studied and consists of an O-linked phosphoryl(C6)-mannose extended with a GlcNAc and a GalNAc. It is formed by sequential action of protein O-mannosyltransferases 1 and 2 (5Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (109) Google Scholar), protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase (6Ogawa M. Nakamura N. Nakayama Y. Kurosaka A. Manya H. Kanagawa M. Endo T. Furukawa K. Okajima T. GTDC2 modifies O-mannosylated alpha-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody.Biochem. Biophys. Res. Commun. 2013; 440: 88-93Crossref PubMed Scopus (26) Google Scholar), β-1,3-N-acetylgalactosaminyltransferase 2 (7Stevens E. Carss K.J. Cirak S. Foley A.R. Torelli S. Willer T. Tambunan D.E. Yau S. Brodd L. Sewry C.A. Feng L. Haliloglu G. Orhan D. Dobyns W.B. Enns G.M. et al.Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of alpha-dystroglycan.Am. J. Hum. Genet. 2013; 92: 354-365Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and protein-O-mannose kinase (8Yoshida-Moriguchi T. Willer T. Anderson M.E. Venzke D. Whyte T. Muntoni F. Lee H. Nelson S.F. Yu L. Campbell K.P. SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function.Science. 2013; 341: 896-899Crossref PubMed Scopus (147) Google Scholar). In the Golgi apparatus, this glycan is further extended by fukutin (FKTN) and fukutin-related protein (FKRP) with a tandem repeat of ribitol phosphate (9Gerin I. Ury B. Breloy I. Bouchet-Seraphin C. Bolsee J. Halbout M. Graff J. Vertommen D. Muccioli G.G. Seta N. Cuisset J.M. Dabaj I. Quijano-Roy S. Grahn A. Van Schaftingen E. et al.ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto alpha-dystroglycan.Nat. Commun. 2016; 7: 11534Crossref PubMed Scopus (73) Google Scholar, 10Kanagawa M. Kobayashi K. Tajiri M. Manya H. Kuga A. Yamaguchi Y. Akasaka-Manya K. Furukawa J.I. Mizuno M. Kawakami H. Shinohara Y. Wada Y. Endo T. Toda T. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy.Cell Rep. 2016; 14: 2209-2223Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 11Praissman J.L. Willer T. Sheikh M.O. Toi A. Chitayat D. Lin Y.Y. Lee H. Stalnaker S.H. Wang S. Prabhakar P.K. Nelson S.F. Stemple D.L. Moore S.A. Moremen K.W. Campbell K.P. et al.The functional O-mannose glycan on alpha-dystroglycan contains a phospho-ribitol primed for matriglycan addition.Elife. 2016; 5e14473Crossref PubMed Scopus (66) Google Scholar). This structure then acts as a scaffold to which the laminin-binding moiety is attached, with multiple repetitions of a disaccharide consisting of xylose and glucuronic acid that are added by the enzymes transmembrane protein 5 (12Manya H. Yamaguchi Y. Kanagawa M. Kobayashi K. Tajiri M. Akasaka-Manya K. Kawakami H. Mizuno M. Wada Y. Toda T. Endo T. The muscular dystrophy gene TMEM5 encodes a ribitol beta1,4-xylosyltransferase required for the functional glycosylation of dystroglycan.J. Biol. Chem. 2016; 291: 24618-24627Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and β-1,4-glucuronyltransferase 1 (13Praissman J.L. Live D.H. Wang S. Ramiah A. Chinoy Z.S. Boons G.J. Moremen K.W. Wells L. B4GAT1 is the priming enzyme for the LARGE-dependent functional glycosylation of alpha-dystroglycan.Elife. 2014; 3e03943Crossref Scopus (62) Google Scholar, 14Willer T. Inamori K. Venzke D. Harvey C. Morgensen G. Hara Y. Beltran Valero de Bernabe D. Yu L. Wright K.M. Campbell K.P. The glucuronyltransferase B4GAT1 is required for initiation of LARGE-mediated alpha-dystroglycan functional glycosylation.Elife. 2014; 3e03941Crossref Scopus (69) Google Scholar) for the first repetition and subsequently by the proteins LARGE1 or LARGE2 (LARGE xylosyl- and glucuronyltransferase 1 or 2) (15Inamori K. Yoshida-Moriguchi T. Hara Y. Anderson M.E. Yu L. Campbell K.P. Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE.Science. 2012; 335: 93-96Crossref PubMed Scopus (199) Google Scholar). FKTN and FKRP in the Golgi apparatus use CDP-ribitol, which is synthesized by the enzyme isoprenoid synthase domain–containing protein in the cytoplasm (9Gerin I. Ury B. Breloy I. Bouchet-Seraphin C. Bolsee J. Halbout M. Graff J. Vertommen D. Muccioli G.G. Seta N. Cuisset J.M. Dabaj I. Quijano-Roy S. Grahn A. Van Schaftingen E. et al.ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto alpha-dystroglycan.Nat. Commun. 2016; 7: 11534Crossref PubMed Scopus (73) Google Scholar, 10Kanagawa M. Kobayashi K. Tajiri M. Manya H. Kuga A. Yamaguchi Y. Akasaka-Manya K. Furukawa J.I. Mizuno M. Kawakami H. Shinohara Y. Wada Y. Endo T. Toda T. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy.Cell Rep. 2016; 14: 2209-2223Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 11Praissman J.L. Willer T. Sheikh M.O. Toi A. Chitayat D. Lin Y.Y. Lee H. Stalnaker S.H. Wang S. Prabhakar P.K. Nelson S.F. Stemple D.L. Moore S.A. Moremen K.W. Campbell K.P. et al.The functional O-mannose glycan on alpha-dystroglycan contains a phospho-ribitol primed for matriglycan addition.Elife. 2016; 5e14473Crossref PubMed Scopus (66) Google Scholar). Yet, the transporter required for the entry of CDP-ribitol into the Golgi remained elusive and is the subject of the presented work. Nucleotide sugars are transported across membranes by specialized proteins known as nucleotide sugar transporters (NSTs) belonging to the SLC35 gene family. These proteins transport nucleotide sugars into the endoplasmic reticulum or the Golgi apparatus while simultaneously exporting the corresponding nucleoside monophosphate (16Hadley B. Maggioni A. Ashikov A. Day C.J. Haselhorst T. Tiralongo J. Structure and function of nucleotide sugar transporters: Current progress.Comput. Struct. Biotechnol. J. 2014; 10: 23-32Crossref PubMed Scopus (65) Google Scholar). Recent structural studies have revealed the concerted conformational changes that are required for this transport (17Ahuja S. Whorton M.R. Structural basis for mammalian nucleotide sugar transport.Elife. 2019; 8e45221Crossref PubMed Scopus (12) Google Scholar). However, the substrate specificity of several transporters is still unknown (18Hadley B. Litfin T. Day C.J. Haselhorst T. Zhou Y. Tiralongo J. Nucleotide sugar transporter SLC35 family structure and function.Comput. Struct. Biotechnol. J. 2019; 17: 1123-1134Crossref PubMed Scopus (14) Google Scholar). Likewise, we are only starting to understand how these transporters distinguish between related substrates (17Ahuja S. Whorton M.R. Structural basis for mammalian nucleotide sugar transport.Elife. 2019; 8e45221Crossref PubMed Scopus (12) Google Scholar, 19Parker J.L. Newstead S. Structural basis of nucleotide sugar transport across the Golgi membrane.Nature. 2017; 551: 521-524Crossref PubMed Scopus (35) Google Scholar). Interestingly, two studies indicated that the Golgi apparatus CMP-sialic acid transporter SLC35A1 could be implicated in CDP-ribitol transport. The first line of evidence comes from a study aiming to identify factors required for the entry of Lassa virus, which is dependent on the interaction with fully glycosylated α-dystroglycan. Using the haploid cell line HAP1, the authors revealed that inactivation of SLC35A1, the CMP-sialic acid transporter, prevented functional glycosylation of α-dystroglycan and Lassa virus entry (20Jae L.T. Raaben M. Riemersma M. van Beusekom E. Blomen V.A. Velds A. Kerkhoven R.M. Carette J.E. Topaloglu H. Meinecke P. Wessels M.W. Lefeber D.J. Whelan S.P. van Bokhoven H. Brummelkamp T.R. Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry.Science. 2013; 340: 479-483Crossref PubMed Scopus (206) Google Scholar). Second, SLC35A1 harboring mutations observed in patients were unable to rescue the α-dystroglycan glycosylation deficiency in SLC35A1 KO HAP1 cell lines (21Riemersma M. Sandrock J. Boltje T.J. Bull C. Heise T. Ashikov A. Adema G.J. van Bokhoven H. Lefeber D.J. Disease mutations in CMP-sialic acid transporter SLC35A1 result in abnormal alpha-dystroglycan O-mannosylation, independent from sialic acid.Hum. Mol. Genet. 2015; 24: 2241-2246Crossref PubMed Scopus (25) Google Scholar). Of note, when sialylation was inhibited by a fluorinated sialic acid analog, α-dystroglycan still bound to its ligand laminin, suggesting that sialylation is not required for functional α-dystroglycan glycosylation. This indicated that the deficiency of α-dystroglycan glycosylation in HAP1 SLC35A1 KO cells was not because of a depletion of CMP-sialic acid in the lumen of the Golgi apparatus (21Riemersma M. Sandrock J. Boltje T.J. Bull C. Heise T. Ashikov A. Adema G.J. van Bokhoven H. Lefeber D.J. Disease mutations in CMP-sialic acid transporter SLC35A1 result in abnormal alpha-dystroglycan O-mannosylation, independent from sialic acid.Hum. Mol. Genet. 2015; 24: 2241-2246Crossref PubMed Scopus (25) Google Scholar). Here, we demonstrate that SLC35A1 and its paralog SLC35A4 play a redundant role in the glycosylation of α-dystroglycan. Using a series of mutants, we reveal that the large binding site of SLC35A1 allows the promiscuous transport of both CMP-sialic acid and CDP-ribitol. Our findings explain why patients with SLC35A1 deficiency do not show clinical symptoms resembling a dystroglycanopathy. To confirm the results obtained by Jae et al. (20Jae L.T. Raaben M. Riemersma M. van Beusekom E. Blomen V.A. Velds A. Kerkhoven R.M. Carette J.E. Topaloglu H. Meinecke P. Wessels M.W. Lefeber D.J. Whelan S.P. van Bokhoven H. Brummelkamp T.R. Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry.Science. 2013; 340: 479-483Crossref PubMed Scopus (206) Google Scholar), we first knocked out SLC35A1 in HAP1 cells using CRISPR/Cas9 and assessed α-dystroglycan glycosylation by flow cytometry using the antibody IIH6 recognizing the functional α-dystroglycan glycan as well as protein sialylation by Arachis hypogaea lectin (peanut agglutinin [PNA]) recognizing nonsialylated Galβ1–3GalNAc and Maackia amurensis lectin II (MAL II), binding to α-2,3-linked sialic acid residues (Fig. 1A). As expected, inactivation of SLC35A1 led to a simultaneous decrease in MAL II staining and increase in PNA staining indicating a general decrease in protein sialylation, which was restored upon re-expression of mSlc35a1 (Fig. 1, B, C, E, and F). SLC35A1 inactivation also led to a decrease in α-dystroglycan glycosylation as measured by flow cytometry using the IIH6 antibody, which was also rescued by re-expression of mSlc35a1 (Fig. 1, D and G). To assess the laminin-binding capacity of α-dystroglycan in SLC35A1 KO cells, we performed a laminin overlay assay (Fig. 1H). The signal intensity (as a surrogate marker for laminin binding) was decreased in SLC35A1 KO cells. In addition, the apparent molecular weight of α-dystroglycan was reduced, likely because of a loss of sialylation and the loss of the laminin-binding glycan. We also observed that the apparent molecular weight of the β-dystroglycan band was reduced, consistent with the known sialylation of this protein. Re-expression of SLC35A1 in KO cell completely rescued the changes in lectin binding (Fig. 1, E and F) and normalized the migration pattern of β-dystroglycan (Fig. 1H, lower panel), consistent with a complete recovery of sialylation because of the known role of SLC35A1 in CMP-sialic acid transport. In contrast, the signal intensity and apparent molecular weight in laminin overlay, as well as the signal in flow cytometry with the antibody IIH6 (Fig. 1, G and H), were increased but did not reach levels observed in wildtype cells. This indicated that the formation of the ribitol phosphate–containing glycan was only partially rescued. Thus, the lower levels of SLC35A1 achieved upon re-expression completely rescued sialylation, whereas higher levels seemed to be required to ensure the formation of the laminin-binding glycan. Our observations could be explained if sialylation was required for the function of a key enzyme involved in the biogenesis of the laminin-binding glycan. However, it has been shown previously that inhibition of sialylation by a synthetic fluorinated sialic acid analog does not affect the biogenesis of the functional glycan (21Riemersma M. Sandrock J. Boltje T.J. Bull C. Heise T. Ashikov A. Adema G.J. van Bokhoven H. Lefeber D.J. Disease mutations in CMP-sialic acid transporter SLC35A1 result in abnormal alpha-dystroglycan O-mannosylation, independent from sialic acid.Hum. Mol. Genet. 2015; 24: 2241-2246Crossref PubMed Scopus (25) Google Scholar). Thus, a more likely explanation is that SLC35A1 not only transports CMP-sialic acid but also CDP-ribitol, albeit potentially with a lower efficiency. Of note, SLC35A1 KO cells still retained some signal in the laminin overlay assay (Fig. 1H) and in flow cytometry with the IIH6 antibody, which recognizes the intact α-dystroglycan glycan (Fig. 1, D and G). This indicates that while SLC35A1 might be the major transporter for CDP-ribitol in HAP1 cells, other transporters might contribute. To quantitatively assess the incorporation of ribitol into α-dystroglycan, we chose human embryonic kidney 293 (HEK293) cells, which we can engineer to produce large amounts of α-dystroglycan secreted in the culture medium. When we knocked out SLC35A1 using CRISPR/Cas9 in these cells, we again observed strong changes in MAL II and PNA lectin flow cytometry, indicating a global deficiency in sialylation (Fig. 2, A and B) . We then purified α-dystroglycan from the culture medium and assessed ribitol incorporation by GC/MS as described previously (9Gerin I. Ury B. Breloy I. Bouchet-Seraphin C. Bolsee J. Halbout M. Graff J. Vertommen D. Muccioli G.G. Seta N. Cuisset J.M. Dabaj I. Quijano-Roy S. Grahn A. Van Schaftingen E. et al.ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto alpha-dystroglycan.Nat. Commun. 2016; 7: 11534Crossref PubMed Scopus (73) Google Scholar). To our surprise, incorporation of ribitol into α-dystroglycan was completely unaffected when SLC35A1 was inactivated in HEK293 cells (Fig. 2C). SLC35A1 was required for the formation of the laminin-binding glycan of α-dystroglycan in HAP1 cells but not required for the incorporation of ribitol into α-dystroglycan in HEK293 cells. This led us to hypothesize that two transporters might play a redundant role in CDP-ribitol transport in HEK293 cells. Assuming that these transporters may belong to the same family, we performed an RT-quantitative PCR analysis of all members of the SLC35A family in HEK293 and HAP1 cells. This revealed that SLC35A4, a family member with largely unknown function (22Sosicka P. Maszczak-Seneczko D. Bazan B. Shauchuk Y. Kaczmarek B. Olczak M. An insight into the orphan nucleotide sugar transporter SLC35A4.Biochim. Biophys. Acta. 2017; 1864: 825-838Crossref PubMed Scopus (14) Google Scholar), was expressed at much higher mRNA levels in HEK293 cells than in HAP1 cells (Fig. 2D). We also reasoned that a candidate CDP-ribitol transporter should be expressed in the brain and muscle, where α-dystroglycan has important functions. Based on RNA-Seq gene expression data obtained from the Genotype-Tissue Expression portal (Fig. S1) (23Carithers L.J. Ardlie K. Barcus M. Branton P.A. Britton A. Buia S.A. Compton C.C. DeLuca D.S. Peter-Demchok J. Gelfand E.T. Guan P. Korzeniewski G.E. Lockhart N.C. Rabiner C.A. Rao A.K. et al.A novel approach to high-quality postmortem tissue procurement: The GTEx project.Biopreserv. Biobank. 2015; 13: 311-319Crossref PubMed Scopus (347) Google Scholar), among SLC35A family members, SLC35A1, and especially SLC35A4 show highest expression levels in skeletal muscle and different brain regions. While SLC35A5 reaches comparable levels in the brain, SLC35A2 and SLC35A3 expression levels were consistently lower. We therefore hypothesized that SLC35A1 and SLC35A4 might play a redundant role in CDP-ribitol transport in HEK293 cells. To test this hypothesis, we proceeded to knock out SLC35A4 in parental HEK293 cells as well as in the previously engineered SLC35A1 KO HEK293 cells. We then purified a secreted α-dystroglycan fragment (aa 1–485) from the supernatant and analyzed ribitol incorporation. This revealed that inactivation of SLC35A4 alone does not significantly affect ribitol incorporation (Fig. 2E). In contrast, inactivation of both SLC35A1 and SLC35A4 simultaneously almost completely abolished ribitol incorporation in the glycan of α-dystroglycan (Fig. 2F). This effect is rescued upon re-expression of either protein in the double KO cells (Fig. 2G), indicating that both proteins independently perform a redundant function in ribitol incorporation into α-dystroglycan. To further investigate the degree of functional redundancy of SLC35A1 and SLC35A4, we went back to HAP1 cells, where we can easily assess endogenous fully glycosylated α-dystroglycan and overall sialylation. We infected SLC35A1 KO HAP1 cells with recombinant lentiviruses driving expression of SLC35A4. Expression of SLC35A4 did not affect general protein sialylation as measured by PNA and MAL II staining using flow cytometry (Fig. 3, A and B). In contrast, we observed that α-dystroglycan glycosylation, assessed by flow cytometry using the IIH6 antibody (Fig. 3C) and laminin overlay (Fig. 3D), was rescued upon SLC35A4 expression. Similar to Figure 1H, we observed again that inactivation of SLC35A1 decreased both binding to laminin and the apparent molecular weight of α-dystroglycan (Figs. 1H and 3D). Interestingly, while SLC35A4 completely rescued the signal in laminin-binding assay to levels exceeding the ones observed in wildtype cells, it only partially rescued the decrease in apparent molecular weight (Fig. 3D, lanes 6 and 8; Fig. 3E) caused by SLC35A1 inactivation. Expression of SLC35A4 did not normalize neither the migration pattern of β-dystroglycan (Fig. 3D) nor lectin binding (Fig. 3, A and B) in SLC35A1 KO cells, suggesting that SLC35A4 does not contribute to CMP-sialic acid transport. This conclusion is also supported by the observation that treatment with sialidase led to a lower apparent molecular weight α- and β-dystroglycan in wildtype and SLC35A1-rescued KO cell lysates, whereas no difference was observed when SLC35A4-expressing SLC35A1 KO cell lysates were treated (Fig. 3E). Thus, we conclude that both transporters are redundant in their contribution to the formation of the laminin-binding glycan, but that only SLC35A1 can transport CMP-sialic acid allowing normal sialylation. Next, we quantified CDP-ribitol and CMP-sialic acid (i.e., CMP-N-acetylneuraminic acid) levels in the SLC35A1 and SLC35A4 single and double KO cells by LC/MS. Inactivation of SLC35A1 led to significant increases in total cellular CMP-sialic acid levels, consistent with the notion that this nucleotide is not efficiently used because of the absence of its transporter (Fig. 3F). Cellular CDP-ribitol levels were unaffected or increased when SLC35A1 and/or SLC35A4 was knocked out. This is consistent with our hypothesis that deficient α-dystroglycan glycosylation in these cell lines is not because of a problem in CDP-ribitol synthesis but rather further downstream. HAP1 cells mainly expressed SLC35A1, whereas HEK293 cells relied on both a redundant function of SLC35A1 and SLC35A4 to assemble the laminin-binding glycan. To test whether a similar redundancy might exist in other settings, we also tested the effect of the depletion of these two proteins in a third cell line. For that purpose, we knocked down both proteins using shRNAs and assessed laminin-binding capacity of α-dystroglycan in HCT116 via laminin overlay (Fig. 3, G and H). Knockdown resulted in a decrease of more than 80% on the mRNA level (Fig. 3I). Single knockdown of either SLC35A1 or SLC35A4 showed no significant effect on the laminin-binding capacity of α-dystroglycan (lanes 2 and 3). However, knocking down both SLC35A1 and SLC35A4 led to a strong decrease of laminin-binding capacity (lane 4). Taken together, our data demonstrate that SLC35A4 and SLC35A1 act in a redundant manner in the assembly of the laminin-binding glycan of α-dystroglycan. Yet, SLC35A4 is not able to overcome the loss of CMP-sialic acid transport in SLC35A1 KO cells, indicating that SLC35A4 cannot transport CMP-sialic acid. One of us recently determined the X-ray crystal structure of the CMP-sialic acid transporter SLC35A1 (17Ahuja S. Whorton M.R. Structural basis for mammalian nucleotide sugar transport.Elife. 2019; 8e45221Crossref PubMed Scopus (12) Google Scholar), which provides a framework for starting to understand the molecular mechanisms of substrate recognition in SLC35A4. There is moderate overall sequence conservation between SLC35A4 and SLC35A1 (24% identity) (Fig. 4); however, nearly all the residues in SLC35A1 that interact with the cytidine group of CMP are conserved in SLC35A4, including Tyr214 and Ser261 (Fig. 4, Fig. S2, and Fig. 5, A and B). We previously proposed that these two residues are critical determinants in conferring nucleotide selectivity since they are Gly and Ala residues, respectively, in the protein products of the SLC35A2 and SLC35A3 genes—the UDP–Gal/GalNAc transporter and the UDP–GlcNAc transporter (NGT), respectively (17Ahuja S. Whorton M.R. Structural basis for mammalian nucleotide sugar transport.Elife. 2019; 8e45221Crossref PubMed Scopus (12) Google Scholar). The fact that these cytidine-interacting residues are conserved between CMP-sialic acid transporter and SLC35A4 suggests that SLC35A4 may be able to selectively transport CMP and other cytidine phosphate–coupled molecules (e.g., CDP-ribitol, CDP-ethanolamine).Figure 5Comparison of substrate-binding sites in SLC35A1 and SLC35A4. A, the structure of CST in complex with CMP-Sia (yellow molecule) is shown. B and C, the structural homology model of SLC35A4 is shown with CDP-ribitol (yellow molecule) computationally docked in either the "compact" (B) or "extended" (C) conformation. In panels A–C, portions of transmembranes (TMs) 1 and 8, which are in front of the substrate, are hidden for clarity. D, a rotated view of the structure of CST in complex with CMP-Sia shown in panel A. E, the same view of the CST structure as in panel D, but with CDP-ribitol computationally docked in. F, a rotated view of what is shown in panel B: the model of SLC35A4 with CDP-ribitol in the compact conformation. In panels D–F, Cα traces are shown for TMs 1 and 7 (instead of cartoon ribbons) for clarity. In panels A–F, key residues are indicated, and select polar interactions are indicated by dashed lines as a visual aid. G, in a view that is similar to that shown in panel D, a surface representation is shown for the CST structure in complex with CMP-Sia. H and I, in a view that is similar to that shown in panel F, a surface repre
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