Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase

• DOI: 10.1016/j.jbc.2021.100769
• Publication Date: 2021-05-08T04:15:00Z
• Authors: Nithya Selvan; Nickita Mehta; Suresh Venkateswaran; Nastry Brignol; Matthew Graziano; M. Osman Sheikh; Yuliya McAnany; Finn Hung; Matthew Madrid; Renee Krampetz; Nicholas Siano; Anuj Mehta; Jon Brudvig; Russell Gotschall; Jill M. Weimer; Hung V. Do
• Keywords: Proteolysis; Sialidase

Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, the deficiency of which leads to Pompe disease. disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but current standard care exhibits poor uptake in skeletal muscles, limiting its clinical efficacy. Furthermore, it unclear how specific cellular processing steps after delivery lysosomes impact undergoes both proteolytic cleavage and glycan trimming within endolysosomal pathway, yielding an that more efficient hydrolyzing natural substrate, glycogen. Here, we developed tool kit modified rhGAAs allowed us dissect individual contributions proteolysis on maturation-associated increases glycogen hydrolysis using vitro cellulo processing, glycopeptide analysis by MS, high-pH anion-exchange chromatography pulsed amperometric detection for kinetics. Chemical modifications terminal sialic acids N-glycans blocked sialidase activity cellulo, thereby preventing downstream without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation as evidenced reduced catalytic efficiency. We also generated enzymatically deglycosylated was shown partially activated despite not undergoing processing. Taken together, these data suggest optimal ERT would require N-glycan attain most treatment Future studies should examine amenability next-generation ERTs types soluble acid hydrolase catabolizes (1Hers H.G. alpha-Glucosidase generalized glycogenstorage (Pompe's disease).Biochem. J. 1963; 86: 11-16Crossref PubMed Scopus (582) Google Scholar). Approximately three-fourths body's total stored muscles (2Jensen Rustad P.I. Kolnes A.J. Lai Y.C. The role muscle breakdown regulation insulin sensitivity exercise.Front. Physiol. 2011; 2: 112Crossref (169) Scholar), unlike cytoplasmic has well-defined biological function energy source, yet been elucidated. Nonetheless, plays essential metabolism help maintain homeostasis many cells, particularly cardiomyocytes cells. Biallelic pathogenic mutations result (glycogen storage type II), phenotypically variable disorder characterized accumulation, progressive weakness, respiratory insufficiency. severe cases have infantile onset typically death 1 year age because cardiac failure (3Kishnani P.S. Hwu W.L. Mandel H. Nicolino M. Yong F. Corzo D. A retrospective, multinational, multicenter study history infantile-onset disease.J. Pediatr. 2006; 148: 671-676Abstract Full Text PDF (412) Scholar, 4van den Hout H.M. Hop W. van Diggelen O.P. Smeitink J.A. Smit G.P. Poll-The B.T. Bakker H.D. Loonen M.C. de Klerk J.B. Reuser der Ploeg A.T. course Pompe's disease: 20 original compared 133 from literature.Pediatrics. 2003; 112: 332-340Crossref (386) Late-onset are low residual milder disease, still significant disability, weakness often leading loss mobility dependence ventilator (5Reuser A.J.J. Hirschhorn R. Kroos M.A. Glycogen II, α-glucosidase (acid maltase) deficiency.in: Valle D.L. Antonarakis S. Ballabio A. Beaudet A.L. Mitchell G.A. Online Metabolic Molecular Basis Inherited Disease. Chapter 135. Part 16. McGraw-Hill, New York, NY2018Google 6Reuser Hermans M.M. Bijvoet A.G. Verbeet M.P. Van Kleijer W.J. Glycogenosis II maltase deficiency).Muscle Nerve Suppl. 1995; 3: S61-S69Crossref (133) patients face prospect early mortality failure. Existing treatments intend restore through (ERT). As other enzymes targeted lysosome, endogenous post-translationally mannose 6-phosphate (M6P), facilitates high-affinity binding cation-dependent M6P receptors cation-independent receptor (CI-MPR), enables transport trans-Golgi network (7Van Bolhuis P.A. Busch Galjaard Receptor-mediated corrects cultured muscle.Pediatr. Res. 1988; 24: 90-94Crossref (36) 8Van Willemsen Brons N.H. Intravenous administration phosphorylated heart mice.J. Clin. Invest. 1991; 87: 513-518Crossref (67) 9Reuser Ponne N.J. Wolterman R.A. H.F. Visser Uptake stability bovine fibroblasts cells glycogenosis patients.Exp. Cell 1984; 155: 178-189Crossref (46) CI-MPR cycles plasma membrane, where endocytosis targeting M6P-bearing exogenous enzymes, including 10Wisselaar H.A. Beeumen Structural functional changes during intracellular maturation.J. Biol. Chem. 1993; 268: 2223-2231Abstract 11McVie-Wylie Lee K.L. Qiu Jin X. Do Gotschall Thurberg B.L. Rogers C. Raben N. O'Callaghan Canfield Andrews L. McPherson J.M. Mattaliano R.J. Biochemical pharmacological characterization different preparations evaluated disease.Mol. Genet. Metab. 2008; 94: 448-455Crossref (70) 12Dahms N.M. Kornfeld receptor. requirements oligomerization.J. 1989; 264: 11458-11467Abstract It this endocytic pathway exploited ERT. unnatural process whereby infused into circulation requires internalized affected target supplement deficient activity. Several factors make difficult rhGAA. problematic issue small fraction intravenously delivered reaches interstitial space such very resultant concentrations (estimated tens nanomolar range) available <1% dose) (13Do H.V. Khanna Challenges treating An industry perspective.Ann. Transl. Med. 2019; 7: 291Crossref therefore critical efficiently via under conditions. However, U.S. Food Drug Administration approved abundance M6P, bisphosphorylated high-mannose (bearing two M6Ps same N-glycan), severely limits at concentrations. Bisphosphorylated known highest affinity all carbohydrate structures enable (12Dahms 14Tong P.Y. Gregory Ligand interactions stoichiometry binding.J. 7962-7969Abstract 1% standard-of-care bear bis-M6P 15Park Kim Y.K. You S.K. Jang Y. Oh D.B. Il H.H. Four unreported glycans containing mannose-6-phosphate heterogeneously attached three sites (including newly found Asn 233) disease.Biochem. Biophys. Commun. 2018; 495: 2418-2424Crossref (9) contains predominantly monophosphorylated approximately 3000-fold lower than (14Tong Because cannot bind concentrations, inefficient Cells defects trafficking surface expression (16Cardone Porto Tarallo Vicinanza Rossi B. Polishchuk E. Donaudy Andria G. De Matteis Parenti Abnormal impairs fibroblasts.Pathogenetics. 1: 6Crossref compounding difficulties pathology widespread time treatment. In limitations, amount exhibit hydrolytic once lysosomes. Endolysosomal appears attaining peak (10Wisselaar 17Bijvoet Pieper F.R. Vliet Boer Recombinant alpha-glucosidase: High level production mouse milk, biochemical characteristics, correction GSDII KO mice.Hum. Mol. 1998; 1815-1824Crossref (73) After delivery, single-chain, 110-kDa precursor sequential mature 76/70 kDa forms (18Moreland Zhang X.K. Decker R.W. Albee Cauthron R.D. Brewer K. Edmunds T. W.M. Lysosomal consists four peptides processed single chain precursor.J. 2005; 280: 6780-6791Abstract (97) Glycan takes place system, monosaccharides sequentially removed resulting five per form opposed 9 11 reported 10-fold higher suggesting events may remove amino or moieties sterically hinder relatively narrow active site secondary carbohydrate-binding domain (19Roig-Zamboni V. Cobucci-Ponzano Iacono Ferrara Germany Bourne Moracci Sulzenbacher Structure alpha-glucosidase-a guide disease.Nat. 2017; 8: 1111Crossref (74) concomitant nature made understand their toward increased Although structural define maturation there reasons believe important. seven potential N-glycosylation sites, six always glycosylated, whereas near C terminus glycosylated 50% One these, 5 (N652), proximal bulky untrimmed here could potentially substrate interactions. individually relative First, chemically block trimming. proceeds manner action exoglycosidases lysosomes, hypothesized blocking residue subsequent sialylated complex-type glycans, assess improving native protein structure effects cleavage. Together, tools discern impacts GAA. To investigate maturation, allow processes independently. N-glycans, abundant (11McVie-Wylie render them nonhydrolyzable. glycerol moiety extending C7 involved recognition exosialidases side-chain O7 O9 oxygens sugar (20Mozzi Mazzacuva P. Zampella Forcella M.E. Fusi Monti insight cytosolic NEU2.Proteins. 2012; 80: 1123-1132Crossref (13) removing oxidation replacing aminooxyacetic (AOAA) disrupt positioning glycosidic bond hydrolysis, exosialidase Given various resident exoglycosidases, reasoned activities impeding access substrates, interrupting impacting nonsialylated Oxidation sodium periodate followed aniline-catalyzed oximation introduce aminooxy groups utilized purpose approach well established applications solid support immobilization tag conjugation (21Zeng Ramya T.N. Dirksen Dawson P.E. Paulson J.C. High-efficiency labeling glycoproteins living cells.Nat. Methods. 2009; 6: 207-209Crossref (285) 22Ramya Weerapana Cravatt B.F. Glycoproteomics enabled tagging acid- galactose-terminated glycans.Glycobiology. 2013; 23: 211-221Crossref (61) 23Hermanson G.T. Bioconjugate Techniques.3th Ed. Academic Press, Cambridge, MA2013Google 2 mM (24Zhu Jiang J.L. Gumlaw N.K. Bercury S.D. Ziegler Kudo Cheng S.H. Glycoengineered improved efficacy correcting metabolic aberrations motor deficits model Ther. 17: 954-963Abstract (100) 25Zhou Q. Stefano J.E. Harrahy Finn Avila Kyazike Wei Patten S.M. Zheng Zhu Pan C.Q. Strategies neoglycan alpha-glucosidase.Bioconjug. 22: 741-751Crossref (33) Scholar) 10 metaperiodate oxidize acid/N-acetylneuraminic (Fig. 1, B). High-pH (HPAEC-PAD) revealed oxidized (∼30%), >90% (data shown). samples reactions AOAA obtain rhGAA–AOAA B) oxime-bonded C7. examined efficiency LC coupled high-resolution tandem MS. unbiased each unmodified greater microheterogeneity previously 26Jongen S.P. Gerwig G.J. Leeflang B.R. Koles Mannesse M.L. Berkel P.H. Zhou Kamerling J.P. expressed milk transgenic rabbits.Glycobiology. 2007; 600-619Crossref (20) although observed were consistent prior reports S1). Examination ∼4 mol added (Table S1, Fig. S2), agreement published reaction efficiencies (25Zhou representative example, analyzed (MS1) higher-energy collisional dissociation MS2 (HCD MS2) spectra tryptic glycopeptide, 882NNTIVNELVR891, bearing highly FA2G2S2 Asn882 [glycan nomenclature according Oxford convention, F indicates core fucose, Ax x number antenna (GlcNAc) trimannosyl complex structure, Gx, linked galactose, Sx, galactose] 2, LC-MS confirmed presence derived 2A). For corresponding rhGAA–AOAA, oxidized, one further 2B). indicate sufficient modification assessing sought generate undergo absence any achieve this, first alpha-mannosidase inhibitor kifunensine (kif) purified secreted referred rhGAA–kif, primarily contained high mannose-type (27Choi H.Y. Park Hong J.K. Kwon J.Y. D.Y. D.I. remodeling mannosidase inhibitors increase rice cell cultures.Sci. Rep. 16130Crossref (12) expected, rhGAA–kif high-mannose-type possible S3). Representative HCD 631ILQFNLLGVPLVGADVCGFLGNTSEE656 652 (site 5), GAA, demonstrate M8 (where Mx represents residues convention) 3, Sites 6 7 contain addition then subjected deglycosylation Endoglycosidase H (Endo H), cleaves between GlcNAc chitobiose leaving (28Freeze Kranz glycoamidase release N-linked glycans.Curr. Protoc. 2010; (Chapter 17, Unit 17.13A)Crossref (14) Endo resulted being abundances S4). notable expected shift retention untreated H–treated glycopeptides (extracted ion chromatogram (EIC) MS1 Figure A–C). MS no occurred purification precursor, peptide coverage obtained termini major advantage able denaturing altering studying direct independent activation. Human sialidases degree specificity 29Smutova Albohy Korchagina Miyagi Bovin Cairo C.W. Pshezhetsky A.V. basis mammalian neuraminidases.PLoS One. 2014; 9e106320Crossref (43) conjugates interfere hydrolysis. whether chemical Sialidase monitored Eastern blotting wheat germ agglutinin (WGA), recognizes acids. Unmodified rhGAA, 30% 90% exhibited robust WGA signal given binds C2 C4 positions pyranose ring (30Gol