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Cited by 36Glycosylation and Glycoproteins ResearchCarbohydrate Chemistry and SynthesisLysosomal Storage Disorders Research

Structure of the immunoregulatory sialidase NEU1

Open access
Gorelik, Alexei, Illes, Katalin, Mazhab-Jafari, Mohammad T., Nagar, Bhushan·2023·Science Advances
DOI 10.1126/sciadv.adf8169PMC PMC10198634
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Abstract

Sialic acids linked to glycoproteins and glycolipids are important mediators of cell and protein recognition events. These sugar residues are removed by neuraminidases (sialidases). Neuraminidase-1 (sialidase-1 or NEU1) is a ubiquitously expressed mammalian sialidase located in lysosomes and on the cell membrane. Because of its modulation of multiple signaling processes, it is a potential therapeutic target for cancers and immune disorders. Genetic defects in NEU1 or in its protective protein cathepsin A (PPCA, CTSA) cause the lysosomal storage diseases sialidosis and galactosialidosis. To further our understanding of this enzyme’s function at the molecular level, we determined the three-dimensional structure of murine NEU1. The enzyme oligomerizes through two self-association interfaces and displays a wide substrate-binding cavity. A catalytic loop adopts an inactive conformation. We propose a mechanism of activation involving a conformational change in this loop upon binding to its protective protein. These findings may facilitate the development of selective inhibitor and agonist therapies.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports the crystal structure of murine neuraminidase-1 (NEU1), revealing a six-bladed β-propeller fold, two self-association interfaces, a wide substrate-binding cavity, and an inactive catalytic-loop conformation. The authors propose that cathepsin A (CTSA) activates NEU1 by inducing a conformational change in this loop.

Key findings

  • The murine NEU1 crystal structure was determined at 3.5 Å resolution; murine NEU1 shares 86% sequence identity with human NEU1 and adopts a six-bladed β-propeller fold with three discernible N-linked glycans and three disulfide bonds.
  • NEU1 oligomerizes through two self-association interfaces: a 650-Ų “beta” interface and an 800-Ų “loop” interface oriented 134° apart; human NEU1 Leu57Asn and Ile249Tyr mutants predominantly remained dimeric by MALS.
  • The active-site loop bearing the catalytic Asp97 adopts an inactive conformation in apo murine NEU1; mutation of the equivalent human Asp103 decreased hydrolysis of 4MU-NANA 100-fold, while Ala106Asn increased isolated human NEU1 activity 4.5-fold but only 50% in the presence of CTSA.
  • NEU1 has a wider substrate-binding cavity than NEU2, explaining why O9-acetylation reduces activity by up to 90% in NEU2, NEU3, and NEU4 but only 50% in NEU1; NEU1 activity was 150-fold higher in the presence than in the absence of CTSA.

Methods

  • Recombinant NEU1 and CTSA were expressed as secreted proteins in Sf9 insect cells; murine NEU1 was crystallized and X-ray diffraction data were collected to 3.5 Å, with the structure solved by molecular replacement using human NEU2 as the search model.
  • Enzymatic activity was measured with 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid (4MU-NANA) at 1 mM, with or without human CTSA; oligomerization was analyzed by SEC-MALS, and NEU1–CTSA interaction was assayed by coprecipitation and SDS-PAGE.
  • Mutants were designed based on the murine NEU1 structure but enzymatic, oligomerization, and coprecipitation assays were carried out with the human homolog.

Limitations

  • The paper states that the 3.5 Å resolution allowed confident backbone and bulky side-chain positioning but many smaller side-chain orientations were modeled approximately; high-resolution crystals of NEU1 with sialic acid were not obtained.
  • The proposed CTSA activation mechanism “remains to be validated through structural studies of the NEU1-CTSA complex,” and only one mutant per self-association interface was tested, so partial overlap between oligomerization and CTSA-binding surfaces cannot be excluded.
  • The text makes evident that membrane association remains unresolved: the crystal structure is incompatible with proposed transmembrane arrangements, and the authors suggest alternative possibilities that “remain to be experimentally investigated.”

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1126/sciadv.adf8169

Cites

90 of 90 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

52 of 76 cited sources checked · 32 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    NEU1 and CTSA can associate into large complexes in the absence of GLB1.

    “Accordingly, NEU1 and CTSA can also associate into large complexes in the absence of GLB1 (21, 22).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [21] A. V. Pshezhetsky, M. Potier, Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of β-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate. J. Biol. Chem. 271, 28359– 28365 (1996). 8910459 source
    • cites [22] E. J. Bonten, A. d'Azzo, Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A. J. Biol. Chem. 275, 37657– 37663 (2000). 10982818 source

      In the Baculovirus Expression in Insect section (page unavailable), neuraminidase formed an approximately 1350-kDa species when co-expressed with PPCA subunits without β-galactosidase (GLB1). But the PPCA subunits were not detected in those high-mass fractions; the authors say their association with neuraminidase may have been transient or below detection. The paper therefore supports large-complex formation without GLB1, but not clearly that both NEU1 and CTSA remain associated in those complexes. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingcitation

    Comparison to human NEU2-DANA confirms that the positions and orientations of the catalytic residues are conserved in NEU1, with the important exception of Asp97.

    “a comparison to the structure of human NEU2 in complex with the sialate-like inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) (11) confirms that the positions and orientations of all these residues are conserved in NEU1 (Fig. 2A), with the important exception of Asp97.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] L. M. Chavas, C. Tringali, P. Fusi, B. Venerando, G. Tettamanti, R. Kato, E. Monti, S. Wakatsuki, Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. J. Biol. Chem. 280, 469– 475 (2005). 15501818 source
  • Premisecitation

    In NEU2, Asp97 is found on a loop that is disordered in the apo protein but points into the active site upon substrate binding.

    “In NEU2, Asp97 is found on a loop that is disordered in the apo protein but points into the active site upon substrate binding (11).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] L. M. Chavas, C. Tringali, P. Fusi, B. Venerando, G. Tettamanti, R. Kato, E. Monti, S. Wakatsuki, Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. J. Biol. Chem. 280, 469– 475 (2005). 15501818 source

      The Neu2-DANA Complex Structure passage (page unavailable) reports that two loops disordered in apo Neu2 become ordered upon inhibitor binding and cover the active site. It identifies Asp46, not Asp97, as the aspartate on one of those loops that moves close to the bound inhibitor. A search for Asp97 found no passage assigning it this role. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    CTSA protects NEU1 from proteolytic degradation.

    “It facilitates the transport of NEU1 to lysosomes (33), protects it from proteolytic degradation (30), and was suggested to induce a conformational change in NEU1, enabling its enzymatic activity (12, 22, 30, 33).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] M. V. Vinogradova, L. Michaud, A. V. Mezentsev, K. E. Lukong, M. El-Alfy, C. R. Morales, M. Potier, A. V. Pshezhetsky, Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation. Biochem. J. 330, 641– 650 (1998). 9480870 PMC1219185 source
  • Premisecitation

    CTSA facilitates the transport of NEU1 to lysosomes.

    “It facilitates the transport of NEU1 to lysosomes (33), protects it from proteolytic degradation (30), and was suggested to induce a conformational change in NEU1, enabling its enzymatic activity (12, 22, 30, 33).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [33] A. van der Spoel, E. Bonten, A. d'Azzo, Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A. EMBO J. 17, 1588– 1597 (1998). 9501080 PMC1170506 source
  • May combine two claims. 37
  • Classified as probably not a substantial claim. 8
  • The quote only partly states this claim. 1

  • May combine two claims.

    Interpretationreasoning

    Leu409 is buried by dimerization and would be inaccessible for sorting.

    “Leu409, which was posited to be part of a YXXΦ sorting motif (17), is buried by dimerization and would be inaccessible for sorting.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] K. E. Lukong, V. Seyrantepe, K. Landry, S. Trudel, A. Ahmad, W. A. Gahl, S. Lefrancois, C. R. Morales, A. V. Pshezhetsky, Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail. J. Biol. Chem. 276, 46172– 46181 (2001). 11571282 source

      The citation is attached to the proposal that Leu409 belongs to a YXXΦ sorting motif, not to the citing authors’ conclusion about dimerization. The source’s Discussion (page not provided) instead identifies the sorting signal as residues 412–415, YGTL, with Leu415—not Leu409—essential for internalization. It does not address whether Leu409 is buried by dimerization. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    NEU1 is part of the GH33 family of glycoside hydrolases.

    “NEU1 is part of the GH33 family of glycoside hydrolases (64), which comprises bacterial and eukaryotic enzymes (65–68) that release terminal sialic acids from glycans or transfer these sugar residues onto other glycans.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [64] E. Drula, M. L. Garron, S. Dogan, V. Lombard, B. Henrissat, N. Terrapon, The carbohydrate-active enzyme database: Functions and literature. Nucleic Acids Res. 50, D571– D577 (2022). 34850161 PMC8728194 source

      The Introduction’s “Maintenance of the modular classification and daily updates” section explains that GH denotes glycoside hydrolase families, but does not identify NEU1 or GH33. Searches for “NEU1 GH33” and “GH33 sialidase neuraminidase” found no passage in our copy establishing NEU1’s family assignment. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    The murine NEU1 crystal structure is most similar to that of human NEU2 and, to a lesser extent, to the Aspergillus fumigatus sialidase.

    “the murine NEU1 crystal structure is most similar to that of human NEU2 (fig. S1A) (11) and, to a lesser extent, to the Aspergillus fumigatus sialidase (67).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [11] L. M. Chavas, C. Tringali, P. Fusi, B. Venerando, G. Tettamanti, R. Kato, E. Monti, S. Wakatsuki, Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. J. Biol. Chem. 280, 469– 475 (2005). 15501818 source
    • cites [67] J. C. Telford, J. H. Yeung, G. Xu, M. J. Kiefel, A. G. Watts, S. Hader, J. Chan, A. J. Bennet, M. M. Moore, G. L. Taylor, The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): Structural and mechanistic insights. J. Biol. Chem. 286, 10783– 10792 (2011). 21247893 PMC3060529 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    NEU1 is a ubiquitously expressed mammalian sialidase located in lysosomes and on the cell membrane.

    “The ubiquitously expressed NEU1 is the most abundant homolog, shares the least sequence similarity with its family members, and is active on oligosaccharides, glycopeptides, and glycoproteins (1, 12–16). It is located in lysosomes and on the cell surface (12, 14, 15, 17, 18).”
    Quote in the paperNot a substantial claim5 of 8 sources checked
    • cites [1] A. V. Pshezhetsky, L. I. Ashmarina, Desialylation of surface receptors as a new dimension in cell signaling. Biochemistry (Mosc.) 78, 736– 745 (2013). 24010837 source
    • cites [12] E. Bonten, A. van der Spoel, M. Fornerod, G. Grosveld, A. d'Azzo, Characterization of human lysosomal neuraminidase defines the molecular basis of the metabolic storage disorder sialidosis. Genes Dev. 10, 3156– 3169 (1996). 8985184 source

      The Expression of human neuraminidase mRNA section reports a transcript in all human tissues tested, and Subcellular localization and enzymatic activation shows lysosome-like distribution. The paper does not establish that this enzyme is on the cell surface; it discusses a plasma-membrane neuraminidase as apparently distinct from the lysosomal enzyme. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [13] C. M. Milner, S. V. Smith, M. B. Carrillo, G. L. Taylor, M. Hollinshead, R. D. Campbell, Identification of a sialidase encoded in the human major histocompatibility complex. J. Biol. Chem. 272, 4549– 4558 (1997). 9020182 source

      The Results (page not available) detect G9 mRNA in several cell types, supporting broad expression but not establishing expression in every tissue. The Abstract and Results report that recombinant G9 localized exclusively to the endoplasmic reticulum in transfected COS7 cells; the paper suggests lysosomal localization as a possibility but does not show G9 in lysosomes or on the cell surface. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [14] A. V. Pshezhetsky, C. Richard, L. Michaud, S. Igdoura, S. Wang, M. A. Elsliger, J. Qu, D. Leclerc, R. Gravel, L. Dallaire, M. Potier, Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis. Nat. Genet. 15, 316– 320 (1997). 9054950 source
    • cites [15] A. V. Pshezhetsky, M. Ashmarina, Lysosomal multienzyme complex: Biochemistry, genetics, and molecular pathophysiology. Prog. Nucleic Acid Res. Mol. Biol. 69, 81– 114 (2001). 11550799 source
    • cites [16] A. d'Azzo, E. Machado, I. Annunziata, Pathogenesis, emerging therapeutic targets and treatment in sialidosis. Expert Opin. Orphan Drugs 3, 491– 504 (2015). 26949572 PMC4777310 source
    • cites [17] K. E. Lukong, V. Seyrantepe, K. Landry, S. Trudel, A. Ahmad, W. A. Gahl, S. Lefrancois, C. R. Morales, A. V. Pshezhetsky, Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail. J. Biol. Chem. 276, 46172– 46181 (2001). 11571282 source

      The Abstract identifies the enzyme as neu-1-encoded sialidase and reports its presence in lysosomes and on the cell surface, particularly in activated T lymphocytes. The passages do not establish that NEU1 is ubiquitously expressed (page numbers are unavailable in our copy). (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [18] A. Hinek, A. V. Pshezhetsky, M. von Itzstein, B. Starcher, Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly. J. Biol. Chem. 281, 3698– 3710 (2006). 16314420 source

  • May combine two claims.

    Premisecitation

    CTSA is sufficient for NEU1 activity, whereas GLB1 is not necessary.

    “However, CTSA is sufficient for NEU1 activity, whereas GLB1 is not necessary (12, 21–24, 27, 32).”
    Quote in the paperNot a substantial claim5 of 7 sources checked
    • cites [12] E. Bonten, A. van der Spoel, M. Fornerod, G. Grosveld, A. d'Azzo, Characterization of human lysosomal neuraminidase defines the molecular basis of the metabolic storage disorder sialidosis. Genes Dev. 10, 3156– 3169 (1996). 8985184 source
    • cites [21] A. V. Pshezhetsky, M. Potier, Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of β-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate. J. Biol. Chem. 271, 28359– 28365 (1996). 8910459 source
    • cites [22] E. J. Bonten, A. d'Azzo, Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A. J. Biol. Chem. 275, 37657– 37663 (2000). 10982818 source
    • cites [23] E. J. Bonten, Y. Campos, V. Zaitsev, A. Nourse, B. Waddell, W. Lewis, G. Taylor, A. d'Azzo, Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization. J. Biol. Chem. 284, 28430– 28441 (2009). 19666471 PMC2788892 source
    • cites [24] G. T. van der Horst, N. J. Galjart, A. d'Azzo, H. Galjaard, F. W. Verheijen, Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta. J. Biol. Chem. 264, 1317– 1322 (1989). 2492018 source
    • cites [27] F. W. Verheijen, S. Palmeri, A. T. Hoogeveen, H. Galjaard, Human placental neuraminidase. Activation, stabilization and association with β-galactosidase and its protective protein. Eur. J. Biochem. 149, 315– 322 (1985). 3922758 source
    • cites [32] A. R. Luu, C. Wong, V. Agrawal, N. Wise, B. Handyside, M. J. Lo, G. Pacheco, J. B. Felix, A. Giaramita, A. d'Azzo, J. Vincelette, S. Bullens, S. Bunting, T. M. Christianson, C. M. Hague, J. H. LeBowitz, G. Yogalingam, Intermittent enzyme replacement therapy with recombinant human β-galactosidase prevents neuraminidase 1 deficiency. J. Biol. Chem. 295, 13556– 13569 (2020). 32727849 PMC7521647 source

  • May combine two claims.

    Findingcitation

    The NEU1 oligomerization mode is unrelated to viral sialidase tetramers, disulfide-mediated NEU3 dimers, or the various lattice interfaces observed in NEU2 crystal structures.

    “The NEU1 oligomerization mode is unrelated to viral sialidase tetramers (62), disulfide-mediated NEU3 dimers (9), or the various lattice interfaces observed in NEU2 crystal structures (11, 75, 76), raising the possibility that NEU1 self-association may be required for complex formation with CTSA.”
    Quote in the paperSubstantial claim2 of 5 sources checked
    • cites [62] J. N. Varghese, W. G. Laver, P. M. Colman, Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution. Nature 303, 35– 40 (1983). 6843658 source
    • cites [9] M. Rodriguez-Walker, J. L. Daniotti, Human Sialidase Neu3 is S-acylated and behaves like an integral membrane protein. Sci. Rep. 7, 4167 (2017). 28646141 PMC5482835 source
    • cites [11] L. M. Chavas, C. Tringali, P. Fusi, B. Venerando, G. Tettamanti, R. Kato, E. Monti, S. Wakatsuki, Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. J. Biol. Chem. 280, 469– 475 (2005). 15501818 source
    • cites [75] L. M. Chavas, R. Kato, N. Suzuki, M. von Itzstein, M. C. Mann, R. J. Thomson, J. C. Dyason, J. McKimm-Breschkin, P. Fusi, C. Tringali, B. Venerando, G. Tettamanti, E. Monti, S. Wakatsuki, Complexity in influenza virus targeted drug design: Interaction with human sialidases. J. Med. Chem. 53, 2998– 3002 (2010). 20222714 source
    • cites [76] S. Buchini, F. X. Gallat, I. R. Greig, J. H. Kim, S. Wakatsuki, L. M. Chavas, S. G. Withers, Tuning mechanism-based inactivators of neuraminidases: Mechanistic and structural insights. Angew. Chem. Int. Ed. Engl. 53, 3382– 3386 (2014). 24591206 source

  • May combine two claims.

    Premisecitation

    Isolated CTSA was dimeric, and isolated NEU1 adopted multiple oligomeric states.

    “Similar to the current results, isolated CTSA was dimeric, and isolated NEU1 adopted multiple oligomeric states.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [23] E. J. Bonten, Y. Campos, V. Zaitsev, A. Nourse, B. Waddell, W. Lewis, G. Taylor, A. d'Azzo, Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization. J. Biol. Chem. 284, 28430– 28441 (2009). 19666471 PMC2788892 source

  • May combine two claims.

    Interpretationown data

    The CTSA homodimer remains intact upon association with NEU1, as is the case in the complex of CTSA with GLB1.

    “These results demonstrate that the CTSA homodimer remains intact upon association with NEU1, as is the case in the complex of CTSA with another lysosomal glycosidase, GLB1 (79).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [79] A. Gorelik, K. Illes, S. M. N. Hasan, B. Nagar, M. T. Mazhab-Jafari, Structure of the murine lysosomal multienzyme complex core. Sci. Adv. 7, eabf4155 (2021). 33980489 PMC8115914 source

  • May combine two claims.

    Premisecitation

    GLB1 is not necessary for NEU1 activity and may compete with NEU1 for access to CTSA.

    “Whereas a third enzyme—the glycosidase GLB1—has often been reported as a member of the high–molecular weight NEU1-CTSA complex (15, 27), GLB1 is not necessary for NEU1 activity (12, 21–24, 27, 32) and may compete with NEU1 for access to CTSA (12, 32).”
    Quote in the paperNot a substantial claim5 of 7 sources checked
    • cites [12] E. Bonten, A. van der Spoel, M. Fornerod, G. Grosveld, A. d'Azzo, Characterization of human lysosomal neuraminidase defines the molecular basis of the metabolic storage disorder sialidosis. Genes Dev. 10, 3156– 3169 (1996). 8985184 source
    • cites [21] A. V. Pshezhetsky, M. Potier, Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of β-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate. J. Biol. Chem. 271, 28359– 28365 (1996). 8910459 source

      In Results and Discussion (page not provided), the paper reports normal NEUR activity in β-galactosidosis fibroblasts lacking the usual complex and says association with CathA alone is sufficient for NEUR activity. This supports GLB1 not being necessary, but the paper does not say that GLB1 may compete with NEU1 for access to CTSA. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [22] E. J. Bonten, A. d'Azzo, Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A. J. Biol. Chem. 275, 37657– 37663 (2000). 10982818 source

      In the Results section (page not available), β-galactosidase/GLB1 had no effect on neuraminidase/NEU1 activity in the insect-cell experiments, supporting the claim that it was not necessary for activation in that setting. The paper also reports that all three proteins can associate, but the passages do not establish that GLB1 competes with NEU1 for access to PPCA/CTSA. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [23] E. J. Bonten, Y. Campos, V. Zaitsev, A. Nourse, B. Waddell, W. Lewis, G. Taylor, A. d'Azzo, Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization. J. Biol. Chem. 284, 28430– 28441 (2009). 19666471 PMC2788892 source

      The Results (page not available) show that purified PPCA activates NEU1 in a cell-free assay without GLB1, supporting that GLB1 is not necessary for activity in that setting. The Discussion (page not available) describes PPCA and NEU1 competing for binding sites on other NEU1 molecules; it does not say that GLB1 competes with NEU1 for access to PPCA. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [24] G. T. van der Horst, N. J. Galjart, A. d'Azzo, H. Galjaard, F. W. Verheijen, Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta. J. Biol. Chem. 264, 1317– 1322 (1989). 2492018 source
    • cites [27] F. W. Verheijen, S. Palmeri, A. T. Hoogeveen, H. Galjaard, Human placental neuraminidase. Activation, stabilization and association with β-galactosidase and its protective protein. Eur. J. Biochem. 149, 315– 322 (1985). 3922758 source
    • cites [32] A. R. Luu, C. Wong, V. Agrawal, N. Wise, B. Handyside, M. J. Lo, G. Pacheco, J. B. Felix, A. Giaramita, A. d'Azzo, J. Vincelette, S. Bullens, S. Bunting, T. M. Christianson, C. M. Hague, J. H. LeBowitz, G. Yogalingam, Intermittent enzyme replacement therapy with recombinant human β-galactosidase prevents neuraminidase 1 deficiency. J. Biol. Chem. 295, 13556– 13569 (2020). 32727849 PMC7521647 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Coincubation of NEU1 and CTSA was reported to produce a 104-kDa heterodimeric complex with one NEU1 and one CTSA copy.

    “Coincubation of both proteins was reported to produce a 104-kDa heterodimeric complex with one NEU1 and one CTSA copy (23), implying that both NEU1 self-association interfaces, as well as the CTSA homodimer, dissociate to allow complex formation.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [23] E. J. Bonten, Y. Campos, V. Zaitsev, A. Nourse, B. Waddell, W. Lewis, G. Taylor, A. d'Azzo, Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization. J. Biol. Chem. 284, 28430– 28441 (2009). 19666471 PMC2788892 source

  • May combine two claims.

    Premisecitation

    Residues responsible for catalysis and stabilization of the sialate carboxyl group are conserved across the sialidase superfamily, while amino acids interacting with the N-acetyl and glycerol moieties are divergent.

    “Although the residues responsible for catalysis and stabilization of the sialate carboxyl group are conserved across the sialidase superfamily, amino acids that interact with the opposite side of the substrate, namely, its N-acetyl and glycerol moieties (Fig. 1B), are divergent (11).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] L. M. Chavas, C. Tringali, P. Fusi, B. Venerando, G. Tettamanti, R. Kato, E. Monti, S. Wakatsuki, Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. J. Biol. Chem. 280, 469– 475 (2005). 15501818 source

  • May combine two claims.

    Premisecitation

    The catalytic loop transitions from disordered to ordered upon substrate binding to NEU2, but in other eukaryotic sialidases of the GH33 family, this loop is productively positioned even without substrate.

    “The corresponding loop transitions from disordered to ordered upon substrate binding to NEU2 (11), but in other eukaryotic sialidases of the GH33 family, this loop is productively positioned even without substrate (65–68).”
    Quote in the paperSubstantial claim3 of 5 sources checked
    • cites [11] L. M. Chavas, C. Tringali, P. Fusi, B. Venerando, G. Tettamanti, R. Kato, E. Monti, S. Wakatsuki, Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. J. Biol. Chem. 280, 469– 475 (2005). 15501818 source
    • cites [65] Y. Luo, S. C. Li, M. Y. Chou, Y. T. Li, M. Luo, The crystal structure of an intramolecular trans-sialidase with a NeuAcα2→3Gal specificity. Structure 6, 521– 530 (1998). 9562562 source

      The Results and discussion (Overall structure; page not provided) report a substrate-free structure of leech IT-sialidase in which residues 81–759 are resolved, and describe a loop in its catalytic domain. This supports the presence of an ordered loop without bound substrate, but the passages do not establish that it is the corresponding catalytic loop or that it is productively positioned. The NEU2 clause is attached to a different citation and is not judged here. (LLM, conf=0.70)

      Experimental — accuracy not yet measured
    • cites [66] A. Buschiazzo, G. A. Tavares, O. Campetella, S. Spinelli, M. L. Cremona, G. París, M. F. Amaya, A. C. Frasch, P. M. Alzari, Structural basis of sialyltransferase activity in trypanosomal Sialidases. EMBO J. 19, 16– 24 (2000). 10619840 PMC1171773 source
    • cites [67] J. C. Telford, J. H. Yeung, G. Xu, M. J. Kiefel, A. G. Watts, S. Hader, J. Chan, A. J. Bennet, M. M. Moore, G. L. Taylor, The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): Structural and mechanistic insights. J. Biol. Chem. 286, 10783– 10792 (2011). 21247893 PMC3060529 source

      The Discussion (page unavailable) says the Aspergillus sialidase active site is very rigid, and Figure 3 compares its substrate-free structure with a ligand-bound sialidase. This supports a preorganized active site, but the paper does not establish that the specific corresponding catalytic loop is productively positioned without substrate. The NEU2 clause is attached to a different citation and is not assessed here. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [68] A. Nejatie, E. Steves, N. Gauthier, J. Baker, J. Nesbitt, S. A. McMahon, V. Oehler, N. J. Thornton, B. Noyovitz, K. Khazaei, B. W. Byers, W. F. Zandberg, T. M. Gloster, M. M. Moore, A. J. Bennet, Kinetic and structural characterization of sialidases (Kdnases) from ascomycete fungal pathogens. ACS Chem. Biol. 16, 2632– 2640 (2021). 34724608 source

  • May combine two claims.

    Interpretationcitation

    The reported 104-kDa heterodimeric complex does not match the large aggregates resulting in coprecipitation of both proteins or the megadalton-size complexes in vivo.

    “This finding does not match the large aggregates resulting in coprecipitation of both proteins described above or the megadalton-size complexes in vivo (21, 22).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [21] A. V. Pshezhetsky, M. Potier, Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of β-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate. J. Biol. Chem. 271, 28359– 28365 (1996). 8910459 source
    • cites [22] E. J. Bonten, A. d'Azzo, Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A. J. Biol. Chem. 275, 37657– 37663 (2000). 10982818 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    NEU1 shares 20 to 21% sequence identity with NEU2, NEU3, and NEU4, which are 41 to 45% identical to each other.

    “NEU1 shares 20 to 21% sequence identity with NEU2, NEU3, and NEU4, which are 41 to 45% identical to each other.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Murine NEU1 contains four potential N-linked glycosylation sites; three glycans are clearly discernible in the structure and are conserved in the human homolog.

    “Murine NEU1 contains four potential N-linked glycosylation sites; three glycans are clearly discernible in the structure and are conserved in the human homolog.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the murine NEU1 structure, residues 139-159 and 316-333 form β strands at the core of the protein, and the C-terminal YGTL peptide does not substantially extend away from the catalytic domain.

    “In the murine NEU1 structure reported here, these residues form β strands at the core of the protein, and the C-terminal YGTL peptide does not substantially extend away from the catalytic domain (Fig. 1A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NEU1 consists of a single domain shaped as a six-bladed β-propeller, with each β sheet composed of four strands.

    “NEU1 consists of a single domain shaped as a six-bladed β-propeller, with each β sheet composed of four strands (Fig. 1A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The loop-substituted mutants formed oligomers in solution as the wild-type and retained a low rate of CTSA-unassisted substrate hydrolysis comparable to wild-type.

    “The mutants formed oligomers in solution as the wild-type (fig. S2, B and C) and retained a low rate of CTSA-unassisted substrate hydrolysis comparable to wild-type (Fig. 4D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In human NEU2, the glycerol moiety of the substrate contacts Glu218, Tyr179, and Tyr181; in murine NEU1, Tyr179 is replaced by His214, Tyr181 by threonine, and Asp257 (Leu in NEU2) establishes two hydrogen bonds with the glycerol group.

    “In human NEU2, the glycerol moiety of the substrate contacts Glu218, Tyr179, and Tyr181, whereas in murine NEU1, Tyr179 is replaced by His214, Tyr181 is replaced by a threonine, and an additional residue (Asp257 that is a leucine in NEU2) establishes two hydrogen bonds with the glycerol group (Fig. 2A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Tyr224Cys CTSA mutant self-dimerizes as the wild-type but cannot dissociate later because of an added covalent Cys244-Cys244 disulfide bond.

    “This mutant self-dimerizes as the wild-type but cannot dissociate later, because of the added covalent Cys244─Cys244 disulfide bond (fig. S4, C, D, and F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ala106Asn increased activity 4.5-fold in isolated human NEU1, but only by 50% in presence of CTSA.

    “Ala106Asn increased activity 4.5-fold in isolated human NEU1, but only by 50% in presence of CTSA (Fig. 4G), supporting this mechanism.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutation of human NEU1 Glu104 did not substantially affect activity with CTSA, whereas mutation of Asp103 decreased hydrolysis of 4MU-NANA 100-fold.

    “Mutation of the corresponding Glu104 in purified human NEU1 did not substantially affect its activity in presence of its activator protein CTSA, whereas mutation of Asp103 (equivalent to murine Asp97) decreased hydrolysis of the small-molecule substrate 100-fold (Fig. 2C).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    An obstacle to the production of recombinant NEU1 is its propensity to aggregate.

    “For example, an obstacle to the production of recombinant NEU1 is its propensity to aggregate;”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The Tyr224Cys CTSA mutant coprecipitated with human NEU1 and activated the sialidase as efficiently as wild-type CTSA.

    “The Tyr224Cys CTSA mutant coprecipitated with human NEU1 (Fig. 4A and fig. S4E) and activated the sialidase as efficiently as wild-type CTSA (Fig. 4B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Wild-type human NEU1 mostly formed high-molecular weight multimers at neutral and acidic pH, whereas the Leu57Asn and Ile249Tyr mutants predominantly remained dimeric.

    “Whereas the wild-type protein mostly formed high–molecular weight multimers at neutral and acidic pH, the mutants predominantly remained dimeric (fig. S3, B to D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At acidic pH, concentrated isolated wild-type NEU1 slowly precipitates, whereas isolated CTSA remains soluble.

    “At acidic pH, concentrated isolated wild-type NEU1 slowly precipitates, whereas isolated CTSA remains soluble.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Purified NEU1 slowly precipitates at acidic pH.

    “Purified NEU1 slowly precipitates at acidic pH, whereas isolated CTSA remains soluble.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The 650-Å2 beta contact surface is formed by a β strand establishing four backbone hydrogen bonds with its counterpart from the adjacent NEU1 monomer and is further stabilized by a small hydrophobic pocket on both sides.

    “The 650-Å2 beta contact surface is formed by a β strand establishing four backbone hydrogen bonds with its counterpart from the adjacent NEU1 monomer and is further stabilized by a small hydrophobic pocket on both sides (Fig. 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The human Ile249Tyr loop interface mutant was inactive in presence of CTSA and did not display the low rate of CTSA-unassisted substrate hydrolysis (1%) observed in wild-type NEU1.

    “Unexpectedly, the human Ile249Tyr loop interface mutant was inactive in presence of CTSA and did not even display the low rate of CTSA-unassisted substrate hydrolysis (1%) observed in wild-type NEU1 (Fig. 3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the apo murine NEU1 structure, the catalytic loop is ordered but folded away from the active site, with Asp97 pointing backward.

    “In contrast, in the apo murine NEU1 structure reported here, the loop is ordered but folded away from the active site, with Asp97 pointing backward (Fig. 2B and fig. S2A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The area around the substrate glycerol and N5-acetyl moieties is more widely open in NEU1 than in NEU2, mainly due to length differences in three loops around the active site.

    “In general, the area around the substrate glycerol and N5-acetyl moieties is more widely open in NEU1 than in NEU2 (Fig. 2D), mainly due to length differences in three loops around the active site which cluster Glu111, Tyr181, and Gln270 around the sialic acid in NEU2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 800-Å2 loop interface is formed by three peripheral loops and is subdivided into a set of polar and charged contacts and a larger hydrophobic surface.

    “The 800-Å2 loop interface is formed by three peripheral loops (distinct from the active site loop bearing Asp97 described earlier) and is subdivided into a set of polar and charged contacts and a larger hydrophobic surface (Fig. 3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Substitution with the NEU3 loop increased baseline activity nearly three-fold, although it is still 40 times lower than that of wild-type NEU1 with CTSA.

    “Notably, substitution with the NEU3 loop increased this baseline activity nearly three-fold, although it is still 40 times lower than that of wild-type NEU1 with CTSA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither of the two NEU1 self-association interfaces is in direct contact with its catalytic or substrate-binding residues, although the loop contact surface is within 13 Å of the putative substrate glycerol moiety.

    “Neither of the two NEU1 self-association interfaces are in direct contact with its catalytic or substrate-binding residues, although the loop contact surface is within 13 Å of the putative substrate glycerol moiety.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Sialidase catalysis proceeds via nucleophilic Tyr364 assisted by Glu258, protonation by Asp97, a covalent tyrosine-sialate intermediate, and hydrolysis by water.

    “The nucleophilic Tyr364, assisted by Glu258, displaces the glycan leaving group, which is protonated by Asp97, resulting in a covalent tyrosine-sialate intermediate. This adduct is then hydrolyzed by water, again with the help of Asp97.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] A. G. Watts, I. Damager, M. L. Amaya, A. Buschiazzo, P. Alzari, A. C. Frasch, S. G. Withers, Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: Tyrosine is the catalytic nucleophile. J. Am. Chem. Soc. 125, 7532– 7533 (2003). 12812490 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Genetic defects in NEU1 or in its protective protein cathepsin A (CTSA) cause the lysosomal storage diseases sialidosis and galactosialidosis.

    “Genetic defects in NEU1 or in its protective protein cathepsin A (PPCA, CTSA) cause the lysosomal storage diseases sialidosis and galactosialidosis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The catalytic loop adopts an inactive arrangement in apo NEU1 and must switch to an active conformation for substrate hydrolysis.

    “This suggests that the loop adopts an inactive arrangement in apo NEU1 and must switch to an active conformation for substrate hydrolysis.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Interpretationown data

    The results support the idea that CTSA induces a conformational change by specifically interacting with the NEU1 catalytic loop.

    “further supporting this mechanism of activation, which remains to be validated through structural studies of the NEU1-CTSA complex.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Most sialidosis mutations are predicted to affect the folding or stability of NEU1, and many others are possibly in this category.

    “Most mutations are predicted to affect the folding or stability of NEU1, and many others are possibly in this category (fig. S1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    The variants Arg78Cys, Arg341Gly, and Tyr370Cys disrupt substrate binding or catalysis directly and may not respond to exogenous CTSA.

    “The variants Arg78Cys, Arg341Gly, and Tyr370Cys disrupt substrate binding or catalysis directly and may not respond to exogenous CTSA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Arg305Cys/Pro and Phe260Tyr are located at the loop self-association interface, supporting the notion that oligomerization may be important for NEU1 stability or function.

    “Last, Arg305Cys/Pro and Phe260Tyr are located at the loop self-association interface, supporting the notion that oligomerization may be important for NEU1 stability or function.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The multienzyme complex likely contains CTSA dimers and NEU1 oligomers.

    “The multienzyme complex forms at acidic pH and likely contains CTSA dimers and NEU1 oligomers, and the contact surface(s) between these two proteins is distinct from their own self-association interfaces.”
    Quote in the paperSubstantial claim
42 of 42

  • The paper relies on this without support.

    Incomplete understanding of NEU1's properties hampers design of selective inhibitors and development of NEU1 and CTSA supplementation therapies.

    “Design of selective NEU1 inhibitors (53, 54) and development of NEU1 and CTSA supplementation therapies, which are at the preclinical stage (35, 55–61), are hampered by an incomplete understanding of the enzyme’s properties.”

  • The paper relies on this without support.

    The murine NEU1 crystal structure is representative of human NEU1 for interpreting human mutations and designing human NEU1 mutants.

    “All point mutations and enzymatic, oligomerization, and coprecipitation assays reported here were carried out with the human homolog based on the murine NEU1 structure.”
2 of 2

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • CTSA mutation and added disulfide have different residue numbers

    Minor

    The CTSA mutation is identified as Tyr224Cys, but the resulting disulfide is identified as Cys244─Cys244. The introduced cysteine cannot be at both positions as stated.

1 of 1

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Authors
Gorelik, A., Illes, K., Mazhab-Jafari, M. T., & Nagar, B.
Affiliations
  • McGill University (CA)
  • Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Corresponding author
Nagar, Bhushan
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2023-05-19
Volume
9
Issue
20
Pages
eadf8169-eadf8169
ISSN
2375-2548 (ISSN-L)
DOI
10.1126/sciadv.adf8169
PMC
PMC10198634
OpenAlex
W4377088110
Cited by
36
References
89
Citations by year
2026: 5 · 2025: 12 · 2024: 17 · 2023: 2
FWCI
3.19
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Science Advances (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
  • U.S. Department of Energy (DE‐ AC02‐06CH11357, AC02‐06CH11357, P30 GM124165, 06CH11357, DE-AC02-)
  • National Institutes of Health (DE-AC02–06CH11357, GM124165)
  • Office of Science (P30 GM124165, AC02‐06CH11357, DE-AC02, 06CH11357, DE-AC02-06CH11357)
  • Canadian Institutes of Health Research
  • National Institute of General Medical Sciences (DE-AC02-06CH11357, GM124165, P30GM124165)
  • Argonne National Laboratory (DE- AC02-06-CH11357, DE-AC02, AC02–06CH11357, 06CH11357, P30 GM124165)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
  • Health Sciences › Medicine › Physiology › Lysosomal Storage Disorders Research
Keywords
NEU1, neuraminidase, sialic acid, enzyme structure, oligomerization, substrate-binding cavity, catalytic loop, conformational change, cathepsin A, sialidosis, galactosialidosis, lysosomal storage disease, enzyme activation mechanism, immune regulation
MeSH terms
Animals, Cell Membrane, Lysosomes, Neuraminidase, Sialic Acids, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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