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• W4309593296 abstract "•The Golgi mannosidase MAN1A1 is phosphorylated at S12 by CDK1 in mitosis•Phosphorylation reduces MAN1A1 activity and affects glycan isomer production•Expression of MAN1A1 phosphorylation mutants alters glycosylation in cells•MAN1A1 mitotic phosphorylation reduces its interaction with glycosyltransferase MGAT1 N-glycans are processed mainly in the Golgi, and a well-organized Golgi structure is required for accurate glycosylation. However, during mitosis the Golgi undergoes severe fragmentation. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes. It is unclear how cells avoid glycosylation defects during mitosis. In this study, we report that Golgi α-1,2-mannosidase IA (MAN1A1), the first enzyme that cargo proteins encounter once arriving the Golgi, is phosphorylated at serine 12 by CDK1 in mitosis, which attenuates its activity, affects the production of glycan isomers, and reduces its interaction with the subsequent glycosyltransferase, MGAT1. Expression of wild-type MAN1A1, but not its phosphomimetic mutant, rescues the glycosylation defects in mannosidase I-deficient cells, whereas expression of its phosphorylation-deficient mutant in mitosis increases the formation of complex glycans. Our study reveals that glycosylation is regulated by cytosolic signaling during the cell cycle. N-glycans are processed mainly in the Golgi, and a well-organized Golgi structure is required for accurate glycosylation. However, during mitosis the Golgi undergoes severe fragmentation. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes. It is unclear how cells avoid glycosylation defects during mitosis. In this study, we report that Golgi α-1,2-mannosidase IA (MAN1A1), the first enzyme that cargo proteins encounter once arriving the Golgi, is phosphorylated at serine 12 by CDK1 in mitosis, which attenuates its activity, affects the production of glycan isomers, and reduces its interaction with the subsequent glycosyltransferase, MGAT1. Expression of wild-type MAN1A1, but not its phosphomimetic mutant, rescues the glycosylation defects in mannosidase I-deficient cells, whereas expression of its phosphorylation-deficient mutant in mitosis increases the formation of complex glycans. Our study reveals that glycosylation is regulated by cytosolic signaling during the cell cycle. IntroductionAsparagine-linked (N-linked) protein glycosylation is a common form of co-translational and post-translational modification of membrane and secreted proteins. The N-glycosylation pathway initiates in the endoplasmic reticulum (ER) with the synthesis and transfer of a pre-formed, high-mannose oligosaccharide precursor to nascent polypeptide chains (Kornfeld and Kornfeld, 1985Kornfeld R. Kornfeld S. Assembly of asparagine-linked oligosaccharides.Annu. Rev. Biochem. 1985; 54: 631-664https://doi.org/10.1146/annurev.bi.54.070185.003215Crossref PubMed Scopus (3754) Google Scholar). After the removal of three glucose residues by the ER glucosidases and one mannose by the ER mannosidase MAN1B1, the oligosaccharide is further processed in the Golgi, first by α-mannosidase I (MAN1A1, MAN1A2, MAN1C1), which removes three mannose residues; followed by α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1; also called GnT1), which adds a GlcNAc residue; Golgi α-mannosidases II (MAN2A1, MAN2A2), which cleaves two terminal mannose residues; and GnT2, which adds another GlcNAc residue. The produced GlcNAcMan3GlcNAc2-Asn core structure is then further processed by branching and capping reactions mediated by other enzymes to form complex type oligosaccharides (Moremen and Nairn, 2014Moremen K.W. Nairn A.V. Mannosidase, alpha, class 1 (MAN1A1 (Golgi alpha-mannnosidase IA), Man1A2 (Golgi alpha-mannosidase IB), MAN1B1(ER alpha-mannosidase I), MAN1C1 (Golgi alpha-mannosidase IC)).in: Taniguchi N. Honke K. Fukuda M. Narimatsu H. Yamaguchi Y. Angata T. Handbook of Glycosyltransferases and Related Genes. Springer, 2014: 1297-1312Crossref Scopus (3) Google Scholar).The Golgi stack consists of an ordered series of compartments that are biochemically and functionally distinct from each other. The cis-Golgi, being closest to the ER from which it receives cargo molecules, contains Golgi MAN1 that removes three mannose residues from the oligosaccharides. The medial-Golgi includes the central layers in the stack where MAN2 removes two mannose residues and several glycosyltransferases add sugars (e.g., GlcNAc) to the glycan chain. The trans-Golgi, which is farthest from the ER, hosts additional glycosyltransferases that add more sugars (e.g., galactose and sialic acid) to the glycoproteins. The trans-Golgi network (TGN), a tubular network extended from the trans-Golgi, sorts cargo molecules for delivery to different destinations. An ordered organization of glycosidases and glycosyltransferases in the subcompartments of the Golgi stack is required for sequential and accurate processing of N-glycans (Zhang and Wang, 2016Zhang X. Wang Y. Glycosylation quality control by the Golgi structure.J. Mol. Biol. 2016; 428: 3183-3193https://doi.org/10.1016/j.jmb.2016.02.030Crossref PubMed Scopus (69) Google Scholar). Disruption of the Golgi structure impairs accurate glycosylation (Puthenveedu et al., 2006Puthenveedu M.A. Bachert C. Puri S. Lanni F. Linstedt A.D. GM130 and GRASP65-dependent lateral cisternal fusion allows uniform Golgi-enzyme distribution.Nat. Cell Biol. 2006; 8: 238-248Crossref PubMed Scopus (253) Google Scholar; Xiang et al., 2013Xiang Y. Zhang X. Nix D.B. Katoh T. Aoki K. Tiemeyer M. Wang Y. Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65.Nat. Commun. 2013; 4: 1659https://doi.org/10.1038/ncomms2669Crossref PubMed Scopus (95) Google Scholar), and glycosylation defects are often linked to Golgi structural disorganization in diseases (Condon et al., 2013Condon K.H. Ho J. Robinson C.G. Hanus C. Ehlers M.D. The Angelman syndrome protein Ube3a/E6AP is required for Golgi acidification and surface protein sialylation.J. Neurosci. 2013; 33: 3799-3814https://doi.org/10.1523/JNEUROSCI.1930-11.2013Crossref PubMed Scopus (30) Google Scholar; Kornak et al., 2008Kornak U. Reynders E. Dimopoulou A. Van Reeuwijk J. Fischer B. Rajab A. Budde B. Nürnberg P. Foulquier F. Lefeber D. et al.ARCL Debré-type Study GroupImpaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2.Nat. Genet. 2008; 40: 32-34https://doi.org/10.1038/ng.2007.45Crossref PubMed Scopus (280) Google Scholar; Percival and Froehner, 2007Percival J.M. Froehner S.C. Golgi complex organization in skeletal muscle: a role for Golgi-mediated glycosylation in muscular dystrophies?.Traffic. 2007; 8: 184-194https://doi.org/10.1111/j.1600-0854.2006.00523.xCrossref PubMed Scopus (25) Google Scholar; D'souza et al., 2020D'souza Z. Taher F.S. Lupashin V.V. Golgi inCOGnito: from vesicle tethering to human disease.Biochim. Biophys. Acta. Gen. Subj. 2020; 1864: 129694https://doi.org/10.1016/j.bbagen.2020.129694Crossref PubMed Scopus (15) Google Scholar).Golgi structure disorganization also occurs under physiological conditions. During mitosis, the Golgi undergoes a series of stepwise disassembly processes controlled by cytoplasmic factors (Huang and Wang, 2017Huang S. Wang Y. Golgi structure formation, function, and post-translational modifications in mammalian cells.F1000Res. 2017; 6: 2050https://doi.org/10.12688/f1000research.11900.1Crossref PubMed Scopus (46) Google Scholar; Tang and Wang, 2013Tang D. Wang Y. Cell cycle regulation of Golgi membrane dynamics.Trends Cell Biol. 2013; 23: 296-304https://doi.org/10.1016/j.tcb.2013.01.008Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar; Wang and Seemann, 2011Wang Y. Seemann J. Golgi biogenesis.Cold Spring Harb. Perspect. Biol. 2011; 3: a005330https://doi.org/10.1101/cshperspect.a005330Crossref PubMed Scopus (50) Google Scholar). A key player is cyclin-dependent kinase 1 (CDK1), which phosphorylates and inactivates multiple Golgi structural proteins including GRASP65 and GM130, leading to mitotic Golgi disassembly (Lowe et al., 1998Lowe M. Rabouille C. Nakamura N. Watson R. Jackman M. Jämsä E. Rahman D. Pappin D.J. Warren G. Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis.Cell. 1998; 94: 783-793Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar; Wang et al., 2003Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. A direct role for GRASP65 as a mitotically regulated Golgi stacking factor.EMBO J. 2003; 22: 3279-3290https://doi.org/10.1093/emboj/cdg317Crossref PubMed Scopus (149) Google Scholar). Previous research suggested that intra-Golgi transport is stopped during mitotic Golgi fragmentation (Collins and Warren, 1992Collins R.N. Warren G. Sphingolipid transport in mitotic HeLa cells.J. Biol. Chem. 1992; 267: 24906-24911Abstract Full Text PDF PubMed Google Scholar). This raises a question: how does the cell avoid inaccurate glycosylation when the cargo molecules and enzymes are trapped together for an extended exposure during mitosis?One possibility is that the activities of glycosylation enzymes are regulated in mitosis. Given that phosphorylation is the driver of cell cycle progression as well as the major cause of Golgi fragmentation, it is possible that Golgi enzymes are also regulated by mitotic kinases. Several Golgi glycosylation enzymes, such as MAN1A1 and MAN1C1 (Bongini et al., 2014Bongini L. Melli L. Lombardi V. Bianco P. Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics.Nucleic Acids Res. 2014; 42: 3436-3449https://doi.org/10.1093/nar/gkt1297Crossref PubMed Scopus (15) Google Scholar), MAN2A1 (Villen et al., 2007Villén J. Beausoleil S.A. Gerber S.A. Gygi S.P. Large-scale phosphorylation analysis of mouse liver.Proc. Natl. Acad. Sci. USA. 2007; 104: 1488-1493https://doi.org/10.1073/pnas.0609836104Crossref PubMed Scopus (623) Google Scholar), and MGAT4A (Tagliabracci et al., 2015Tagliabracci V.S. Wiley S.E. Guo X. Kinch L.N. Durrant E. Wen J. Xiao J. Cui J. Nguyen K.B. Engel J.L. et al.A single kinase generates the majority of the secreted phosphoproteome.Cell. 2015; 161: 1619-1632https://doi.org/10.1016/j.cell.2015.05.028Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), have been identified in phosphoproteomic studies of interphase cells. However, there is so far no report on cell cycle-dependent phosphorylation or regulation of Golgi glycosylation enzymes.Here, we performed phosphoproteomic studies of mitotic Golgi membranes and discovered that MAN1A1 is highly phosphorylated at serine 12 (S12) at its cytoplasmic domain by CDK1 in mitosis, which inhibits MAN1A1 activity through reducing its interaction with the subsequent glycosylation enzyme MGAT1.Our study reveals that glycosylation is regulated by phosphorylation in the cell cycle.ResultsPhosphoproteomic analysis reveals that MAN1A1 is highly phosphorylated in mitosisTo identify candidate proteins regulated by phosphorylation during the cell cycle, we purified interphase Golgi membranes from rat liver (RLG), prepared mitotic Golgi fragments (MGFs) by incubating RLG with mitotic cytosol (Tang and Wang, 2015Tang D. Wang Y. Golgi isolation.Cold Spring Harb. Protoc. 2015; 2015: 562-567https://doi.org/10.1101/pdb.prot075911Crossref PubMed Scopus (2) Google Scholar; Wang et al., 2006Wang Y. Taguchi T. Warren G. Purification of rat liver Golgi stacks.in: Celis J. Cell Biology: A Laboratory Handbook. Third Edition. Elsevier Science, 2006: 33-39Crossref Scopus (13) Google Scholar; Tang et al., 2010Tang D. Xiang Y. Wang Y. Reconstitution of the cell cycle-regulated Golgi disassembly and reassembly in a cell-free system.Nat. Protoc. 2010; 5: 758-772https://doi.org/10.1038/nprot.2010.38Crossref PubMed Scopus (31) Google Scholar), and performed mass spectrometry (MS) analysis to identify phosphorylated proteins (Kweon and Andrews, 2013Kweon H.K. Andrews P.C. Quantitative analysis of global phosphorylation changes with high-resolution tandem mass spectrometry and stable isotopic labeling.Methods. 2013; 61: 251-259https://doi.org/10.1016/j.ymeth.2013.04.010Crossref PubMed Scopus (10) Google Scholar). The results revealed previously reported mitotic phosphorylation of Golgi structure proteins, including GM130 (Lowe et al., 2000Lowe M. Gonatas N.K. Warren G. The mitotic phosphorylation cycle of the cis-Golgi matrix protein GM130.J. Cell Biol. 2000; 149: 341-356Crossref PubMed Scopus (126) Google Scholar), GRASP65 (Tang et al., 2012Tang D. Yuan H. Vielemeyer O. Perez F. Wang Y. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly.Biol. Open. 2012; 1: 1204-1214https://doi.org/10.1242/bio.20122659BIO20122659Crossref PubMed Google Scholar), Giantin (Dephoure et al., 2008Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. USA. 2008; 105: 10762-10767https://doi.org/10.1073/pnas.0805139105Crossref PubMed Scopus (1235) Google Scholar; Olsen et al., 2010Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. et al.Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3https://doi.org/10.1126/scisignal.2000475Crossref PubMed Scopus (1128) Google Scholar), and Golgin 84 (Diao et al., 2003Diao A. Rahman D. Pappin D.J.C. Lucocq J. Lowe M. The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation.J. Cell Biol. 2003; 160: 201-212Crossref PubMed Scopus (196) Google Scholar; Dephoure et al., 2008Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. USA. 2008; 105: 10762-10767https://doi.org/10.1073/pnas.0805139105Crossref PubMed Scopus (1235) Google Scholar) (Table S1), as well as a number of Golgi enzymes, including MAN1A1, MAN1A2, ST6GAL1, and GALNT11 (Figure 1A ; Table S1). Among these, serine 12 on MAN1A1 was most highly phosphorylated in mitosis than interphase, while S11 on MAN1A1 was phosphorylated at a very low abundance in only one of the two MS results and was not specific to mitosis (Table S1) and thus was not a focus in this study. In addition, threonine 2 (T2), threonine 3 (T3), and serine 10 (S10) on MAN1A2 were also phosphorylated (Figure 1A).Alignment of MAN1A1 amino acid sequences showed that S12 is highly conserved across mammalian species where mitotic Golgi fragmentation has been documented (Figure 1B). To confirm MAN1A1 phosphorylation in mitotic cells, we enriched mitotic cells by nocodazole synchronization (Tang et al., 2012Tang D. Yuan H. Vielemeyer O. Perez F. Wang Y. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly.Biol. Open. 2012; 1: 1204-1214https://doi.org/10.1242/bio.20122659BIO20122659Crossref PubMed Google Scholar), immunoprecipitated MAN1A1 from mitotic and interphase cells, and determined its phosphorylation by western blot using a phospho-serine (p-Ser) antibody. The result showed that MAN1A1 was phosphorylated in mitotic but not interphase cells (Figure 1C). Similarly, T2, T3, and S10 on MAN1A2 are also conserved among species (Figure S1A), and mitotic phosphorylation of MAN1A2 was confirmed using a phospho-threonine (p-Thr) antibody (Figure S1B). As a negative control, MAN2A1 was not phosphorylated in our phosphoproteomic and biochemical analyses (Figure S1C). Moreover, phosphorylation of both endogenous and exogenous MAN1A1 in mitosis was confirmed by phos-tag gel analysis (Figures 1D and S1D). The same experiment also revealed that phosphorylation of MAN1A1 is specific to mitosis (indicated by cyclin B1 expression) and is reversible; the upshifted MAN1A1 band in mitosis was downshifted when the cells were released into interphase by nocodazole washout (Figures 1D and S1D). Taken together, MAN1A1 is highly phosphorylated in mitosis but not interphase.MAN1A1 is phosphorylated at S12 by CDK1Given that S12 in MAN1A1 fits the S/T-P consensus sequence for CDK1 phosphorylation, we tested the possibility that MAN1A1 might be phosphorylated by CDK1. We treated mitotic HeLa cells with a highly selective CDK1 inhibitor, RO-3306, a more general CDK inhibitor, roscovitine, or a general kinase inhibitor, staurosporine, and analyzed MAN1A1 phosphorylation by phos-tag gel electrophoresis and western blot. As shown in Figures 2A and 2B , inhibition of CDK1 effectively abolished mitotic phosphorylation of MAN1A1 as indicated by the band-shift on the phos-tag gel. In these experiments, cyclin B protein level remained high after CDK1 inhibition (Figure 2A, lanes 3–7; Figure 2B, lane 3), indicating that such a short-term CDK1 inhibition did not lead to mitotic exit.Figure 2MAN1A1 is phosphorylated at S12 by CDK1Show full caption(A) MAN1A1 is phosphorylated by CDK1 in mitotic (Mit) but not interphase (Int) cells.(B) Endogenous MAN1A1 is phosphorylated by CDK1 in mitotic cells. RO-3306, 10 μM for 30 min.(C) Mutation of S12 abolishes MAN1A1 phosphorylation in mitotic cells.(D) MAN1A1 is phosphorylated in mitotic Golgi fragments (MGF) but not interphase Golgi membranes (RLG). p-GRASP65, GRASP65 was blotted with a phospho-specific antibody.(E) In vitro phosphorylation of MAN1A1 by CDK1 in purified Golgi membranes. Purified RLG membranes were incubated with mitotic cytosol (MGF) or with purified cyclin B1/CDK1 (7.5, 15 or 30 μg in lanes 3–5) and analyzed using western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further specify that S12 of MAN1A1 is phosphorylated in mitosis, we generated the phospho-deficient S12A and phosphomimetic S12E mutants. When expressed in cells, only wild-type (WT) MAN1A1, but not its mutants, was phosphorylated in mitotic cells (Figure 2C). Phosphorylation of WT MAN1A1 was inhibited by RO-3306, whereas S12A and S12E were not affected (Figure S2). These results demonstrate that MAN1A1 is phosphorylated at S12 by CDK1 in mitosis.We then confirmed that MAN1A1 phosphorylation occurs on purified mitotic but not interphase Golgi membranes (Figure 2D). Further treatment of MGFs with calf intestinal alkaline phosphatase (CIP) reversed MAN1A1 phosphorylation, and the effect of CIP was inhibited by β-glycerophosphate, a general phosphatase inhibitor (Figure 2D). To verify that MAN1A1 is directly phosphorylated by CDK1 on Golgi membranes, we treated purified Golgi membranes with purified CDK1 (cyclin B1/CDK1 protein complex), which effectively phosphorylated MAN1A1, as indicated by a similar shift of the MAN1A1 band by CDK1 and by mitotic cytosol (Figure 2E, lanes 3–5 versus 2). In comparison with MAN1A1, CDK1 was less effective in phosphorylating GRASP65, which is consistent with previous reports that GRASP65 phosphorylation requires both CDK1 and Plk1 (Preisinger et al., 2005Preisinger C. Körner R. Wind M. Lehmann W.D. Kopajtich R. Barr F.A. Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a mechanism for Golgi checkpoint signalling.EMBO J. 2005; 24: 753-765Crossref PubMed Scopus (128) Google Scholar; Wang et al., 2003Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. A direct role for GRASP65 as a mitotically regulated Golgi stacking factor.EMBO J. 2003; 22: 3279-3290https://doi.org/10.1093/emboj/cdg317Crossref PubMed Scopus (149) Google Scholar, Wang et al., 2005Wang Y. Satoh A. Warren G. Mapping the functional domains of the Golgi stacking factor GRASP65.J. Biol. Chem. 2005; 280: 4921-4928Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In conclusion, MAN1A1 is phosphorylated at S12 by CDK1 on mitotic Golgi membranes.MAN1A1 phosphorylation inhibits its activityIn the Golgi, mannose residues on the Man8 isomer (Man8B) are cleaved by a combination of MAN1A1, MAN1A2, and MAN1C1 to trim high-mannose oligosaccharide chains to the Man5 isomer (Figure 3A ; also see Figure 4A ). To determine whether MAN1A1 phosphorylation affects its enzymatic activity, we performed a Golgi MAN1 assay using RLG and MGFs. In brief, purified RLG or MGF membranes were lysed and incubated with a mannosidase glycan substrate, pyridylaminated Man9GlcNAc2 (Man9-PA). In this reaction, the long mannose chain of the Man9-PA substrate was trimmed to shorter chains (Man8, Man7, Man6, and Man5) which were distinguished and quantified on the basis of the retention time on high-performance liquid chromatography (HPLC) (Figure 3B). The result indicated that MGFs exhibited lower MAN1 activity than RLG, as indicated by the reduction of the trimmed mannose index (Figure 3C). A time course incubation showed that MGFs exhibited consistently lower MAN1 activity than RLG under different incubation times (Figures S3A–S3E). Meanwhile, Golgi MAN2 activity showed no difference between interphase and mitosis (Figures 3D and S3F–S3H).Figure 3Mitotic phosphorylation reduces Golgi MAN1 activityShow full caption(A) A schematic of the N-glycosylation pathway.(B) Mitotic Golgi fragments (MGF) exhibit reduced MAN1 activity than interphase Golgi membranes (RLG). Shown are representative HPLC profiles from 3 independent experiments after 1 h incubation. Note the reduced MAN1 activity that trims long mannose chains (peaks on the right) to shorter chains (peaks on the left) in MGF (blue line).(C) Trimmed mannose index shows reduced MAN1 activity in MGF. Results are expressed as mean ± SD from three independent experiments.(D) MAN2 activity does not change in mitosis.(E) MAN1 activity in Golgi membranes is regulated by phosphorylation and dephosphorylation.(F) MAN1A1 activity is regulated by CDK1-dependent phosphorylation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Mitotic phosphorylation of Golgi membranes alters the production of different isomers of glycansShow full caption(A) A schematic diagram of the branched Man9-PA substrate including the residue nomenclature and glycosidic linkages for mannose and N-acetylglucosamine residues, and the produced Man8-Man5 isomers.(B–D) Mitotic phosphorylation of MAN1A1 alters the production of Man8, Man7, and Man6 isomers. Shown are the dual-gradient, reversed-phase HPLC profiles.(E) Analyses of the trimming activity of each branch on the basis of the production of all isomers from (B)–(D). Results are expressed as mean ± SD from three independent experiments. The p value was determined using Student’s t test. ∗p < 0.05.(F) Established substrate specificities for MAN1B1, MAN1A1, and MAN1A2 for the digestion series of Man9 to Man5. All predicted isomer intermediates for Man8, Man7, and Man6 are displayed, and MAN1B1 actions are shown in red.(G) The abundances of the Man8, Man7, and Man6 isomer intermediates were determined for the 30 min digestion of Man9-PA by the RLG membrane preparation on the basis of dual-gradient, reversed-phase HPLC (Figure S4A). Values for each isomer abundance in its respective size fraction were normalized as a percentage of total for that size fraction. Results represent mean ± SD from triplicate analyses (red bars). The data were then modeled on the basis of the established substrate specificities of MAN1B1, MAN1A1, and MAN1A2 (Figure 4F), and the abundances of the three enzymes were adjusted to result in a modeled dataset (tan bars) that best matched the profiles of the respective experimental isomer intermediates.(H) The abundances of the Man8, Man7, and Man6 isomer intermediates were determined for the 30 min digestion of Man9-PA by the MGF membrane preparation as in (G). Results represent mean ± SD from triplicate analyses (blue bars). The data were then modeled as in (F) and the abundances of the three enzymes were adjusted to result in a modeled dataset (tan bars) that best matched the profiles of the respective experimental isomer intermediates.(I) The modeled activities of MAN1B1, MAN1A1, and MAN1A2 for 30 min digestion of Man9-PA by RLG (G) and MGF (H) fractions were used to display changes in relative enzyme activities in the two membrane fractions. It was assumed that MAN1B1 activity did not change between the two membrane fractions, and the activities of MAN1A1 and MAN1A2 were then plotted relative to MAN1B1 to determine their respective changes between RLG and MGF.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then confirmed that the reduced MAN1 activity in mitotic Golgi membranes was due to phosphorylation, as dephosphorylation of MAN1A1 with CIP (Figure 2D) recovered MAN1 activity, whereas inhibition of CIP by β-glycerophosphate failed to do so (Figure 3E). Reversely, phosphorylation of MAN1A1 by purified CDK1 reduced MAN1 activity (Figure 3F). In addition, MAN1 activity was not affected by swainsonine, a MAN2 inhibitor (Figure S3I), indicating high specificity of the assay. Taken together, mitotic phosphorylation inhibits MAN1A1 activity.MAN1A1 phosphorylation affects the production of different isomers of glycansWhen examining the peaks of the different oligosaccharide products in our MAN1 activity assay, we noticed that mitotic phosphorylation affected not only the levels of the peaks but also the sub-peaks that represent different isomers within the same length oligosaccharides, such as Man8, Man7, and Man6 (Figure 3B). There are three Man8 isomers that can be generated following mannosidase digestion, Man8A, Man8B, and Man8C, depending on which branch is trimmed on Man9 (Xiang et al., 2016Xiang Y. Karaveg K. Moremen K.W. Substrate recognition and catalysis by GH47 alpha-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway.Proc. Natl. Acad. Sci. USA. 2016; 113: E7890-E7899https://doi.org/10.1073/pnas.1611213113Crossref PubMed Scopus (19) Google Scholar). Similarly, four Man7 isomers and three Man6 isomers can be produced during trimming to the Man5 processing intermediate (Figure 4A) (Lal et al., 1998Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Substrate specificities of recombinant murine Golgi alpha1, 2-mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1, 2-mannosidases.Glycobiology. 1998; 8: 981-995https://doi.org/10.1093/glycob/8.10.981Crossref PubMed Scopus (103) Google Scholar). These isomers were detected as sub-peaks on the HPLC profiles (Figure 3B), and the changes in the sub-peaks indicate that mitotic phosphorylation may affect MAN1A1 activity differently on different branches. Therefore, we further analyzed the glycan isomers by dual-gradient, reversed-phase HPLC (Figures 4B–4D). At 30 min, while interphase Golgi produced mainly Man8A and a lesser amount of Man8B (54.2% and 30.8% of total Man8, respectively, calculated on the basis of the results shown in Figure S4A), as previously reported (Lal et al., 1998Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Substrate specificities of recombinant murine Golgi alpha1, 2-mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1, 2-mannosidases.Glycobiology. 1998; 8: 981-995https://doi.org/10.1093/glycob/8.10.981Crossref PubMed Scopus (103) Google Scholar), mitotic Golgi produced Man8A and Man8B in the opposite ratio (27.7% Man8A and 59.9% Man8B) (Figure S4A). Similar results were obtained at 60 min incubation (Figures S4A and S4B). When considering all products, interphase Golgi exhibited more activity to trim the A- and C-branches, which were inhibited by mitotic phosphorylation (Figure 4E). These results demonstrate that MAN1A1 phosphorylation affects the production of different isomers of glycans.Our previous quantitative proteomic studies demonstrated that within the 4 forms of MAN1 (MAN1A1, MAN1A2, MAN1B1, and MAN1C1), MAN1A1 is the most abundant in purified Golgi membranes, with the ratio of MAN1A1, MAN1A2 and MAN1B1 approximately 2:1:1 (to be more accurate, 27:14:12) according to the peptides detected, whereas MAN1C1 was not detected and so might have a low abundance in Golgi membranes (Chen et al., 2010Chen X. Simon E.S. Xiang Y. Kachman M. Andrews P.C. Wang Y. Quantitative proteomics analysis of cell cycle-regulated Golgi disassembly and reassembly.J. Biol. Chem. 2010; 285: 7197-7207https://doi.org/10.1074/jbc.M109.047084Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Of note, MAN1B1, which exclusively produces the Man8B isomer in the ER and Golgi, is also found in the isolated Golgi membrane fractions. Although the abundance of the MAN1A1, MAN1A2, and MAN1B1 proteins does not change during mitosis (Figures 1C and 12; also see Figure 7) (Chen et al., 2010Chen X. Simon E.S. Xiang Y. Kachman M. Andrews P.C. Wang Y. Quantitative proteomics analysis of cell cycle-regulated Golgi disassembly and reassembly.J. Biol. Chem. 2010; 285: 7197-7207https://doi.org/10.1074/jbc.M109.047084Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), the differences in glycan isomer abundance during in vitro digestions of Man9 between RLG and MGF membranes indicate that the respective enzyme activities are altered during mitosis.Previous studies have determined the respective substrate specificities and isomer profiles for MAN1B1, MAN1A1, and MAN1A2 in the" @default.
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• W4309593296 title "Mitotic phosphorylation inhibits the Golgi mannosidase MAN1A1" @default.
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