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WO2001021769A2 - STRUCTURE TRIDIMENSIONNELLE ET CRISTAL D'UNE α1,2-MANNOSIDASE DE CLASSE I, ET PROCEDES D'UTILISATION DE CES SUBSTANCES - Google Patents

STRUCTURE TRIDIMENSIONNELLE ET CRISTAL D'UNE α1,2-MANNOSIDASE DE CLASSE I, ET PROCEDES D'UTILISATION DE CES SUBSTANCES Download PDF

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Publication number
WO2001021769A2
WO2001021769A2 PCT/CA2000/001093 CA0001093W WO0121769A2 WO 2001021769 A2 WO2001021769 A2 WO 2001021769A2 CA 0001093 W CA0001093 W CA 0001093W WO 0121769 A2 WO0121769 A2 WO 0121769A2
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Prior art keywords
mannosidase
dimensional structure
enzyme
crystal
class
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PCT/CA2000/001093
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English (en)
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WO2001021769A3 (fr
Inventor
Annette Herscovics
Francesco Lipari
Barry Sleno
Lynne P. Howell
François VALLÉE
Pedro A. Romero
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Mcgill University
The Hospital For Sick Children
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Priority to AU73989/00A priority Critical patent/AU7398900A/en
Publication of WO2001021769A2 publication Critical patent/WO2001021769A2/fr
Publication of WO2001021769A3 publication Critical patent/WO2001021769A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01113—Mannosyl-oligosaccharide 1,2-alpha-mannosidase (3.2.1.113), i.e. alpha-1,2-mannosidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases

Definitions

  • the present invention relates to a three- dimensional structure for endoplasmic reticulum (ER) ⁇ - annosidase, more particularly that of the endoplasmic reticulum ⁇ l, 2-mannosidase enzyme family, to a crystal and to methods of use thereof,
  • ER endoplasmic reticulum
  • 2-mannosidase enzyme family a three- dimensional structure for endoplasmic reticulum (ER) ⁇ - annosidase, more particularly that of the endoplasmic reticulum ⁇ l, 2-mannosidase enzyme family, to a crystal and to methods of use thereof
  • ER endoplasmic reticulum
  • ⁇ - annosidase enzyme family
  • Class I ⁇ -mannosidases specifically hydrolyze ⁇ l, 2 -linked mannose residues, and do not cleave substrates such as p-nitrophenyl- ⁇ -D- mannopyranoside . They require calcium for activity and are inhibited by 1-deoxymannoj irimycin and kifunensine, but not by swainsonine .
  • N-glycan formation begins with the transfer of a preformed oligosaccharide precursor, usually Glc 3 Man 9 GlcNAc 2 , to nascent polypeptide chains.
  • the oligosaccharide precursor is immediately trimmed by ⁇ -glucosidases and ⁇ - mannosidases in the endoplasmic reticulum (ER) .
  • Glycoproteins that have acquired their native conformation can then be transported to the Golgi apparatus, where additional ⁇ -mannosidases produce the appropriate substrates for Golgi glycosyltransferases to form the variety of biologically important oligosaccharide structures found on glycoproteins (A. Varki, Glycobiology 3 , 97 (1993)).
  • ER processing glycosidases also play a role in quality control, ensuring that only properly folded proteins are transported to their final destination. Trimming of the oligosaccharide precursor by ⁇ -glucosidase I and II controls the interaction of newly-formed glycoproteins with the lectin chaperones, calnexin and calreticulin, thus facilitating folding of glycoproteins (C.Hammond and A. Helenius, Curr. Opin. Cell Biol. 7, 523 (1995) ) , while trimming of mannose residues in the ER acts as a signal to target misfolded glycoproteins for degradation by the proteasome (K. Su, T. Stoller, J.
  • Saccharomyces cerevisiae there is only one processing ⁇ -mannosidase (Swiss Prot accession number P32906) .
  • This enzyme is a 63kDa type II ER transmembrane glycoprotein with no significant cytoplasmic tail, an N-terminal transmembrane domain
  • yeast and human endoplasmic reticulum (ER) ⁇ l , 2 -mannosidases are highly specific and trim 20 Man 9 GlcNAc 2 to Man 8 GlcNAc 2 isomer B, while mammalian
  • Man 5 GlcNAc 2 The yeast ⁇ l , 2-mannosidase is extremely specific and removes a single mannose residue from
  • Man 9 GlcNAc 2 to form Man 8 GlcNAc 2 isomer B J. C. Byrd, A. 25 . Tarentino, F. Maley, P. H. Atkinson, R. B. Trimble,
  • This processing ⁇ l , 2 - mannosidase may therefore have an important role in genetic diseases characterized by rapid degradation of misfolded glycoproteins such as cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis and ⁇ l-antitrypsin in emphysema (R. N. Sifers, Nat. Struct. Biol. 2, 355 (1995)) .
  • CFTR cystic fibrosis transmembrane conductance regulator
  • emphysema R. N. Sifers, Nat. Struct. Biol. 2, 355 (1995)
  • the action of the ER ⁇ l , 2 -mannosidases in both yeast and mammalian cells triggers the degradation of misfolded glycoproteins.
  • One aim of the present invention is to provide three-dimensional structures and crystals for endoplasmic reticulum (ER) ⁇ l , 2 -mannosidase enzymes, and more particularly detailed three-dimensional structural information for the ER ⁇ l , 2 -mannosidase enzyme family.
  • Another aim of the present invention is to provide identification of structural determinants responsible for the specificity of the different enzymes of the ⁇ l , 2 -mannosidase enzyme family.
  • Yet another aim of the present invention is to provide methods to develop drugs such as agonist, antagonist or inhibitors specific for the ER ⁇ l , 2 - mannosidase enzyme family, which may be used to develop drugs to stabilize abnormal, misfolded glycoproteins in genetic diseases, including, without limitations, cystic fibrosis and pulmonary emphysema.
  • the agonist or antagonist may activate or inhibit the activity of the enzyme for a transient period of time, preventing or activating degradation of misfolded, abnormal glycoproteins.
  • the yeast ER ⁇ l , 2 -mannosidase is the first member of the Class I ⁇ l , 2 -mannosidases whose three- - 7 -
  • the three-dimensional structure of the yeast ⁇ l , 2 -mannosidase of the present invention may be used to deduce that of the enzyme of other species , such as mammalians , and more particularly the human.
  • drugs may be developed to control genetic diseases caused by glycoprotein misfolding including cystic fibrosis and emphysema , such as by computer analyses with a computer program that analyzes molecular structure and interactions .
  • a crystal of a protein- ol igosaccharide/carbohydrate complex comprising a catalytic domain of a class I ⁇ l , 2 -mannosidase enzyme trimming Man 9 GlcNAc 2 to Man 8 GlcNAc 2 isomer B , said crystal ef fectively diffracting X-rays at a resolution of about 1 . 54 Angstroms , thus providing the first detailed three-dimensional structure of a Class I ⁇ l,2- mannosidase .
  • the ⁇ l , 2 -mannosidase may be derived from a yeast such as Saccharomyces cerevisiae .
  • the ⁇ l , 2 - mannosidase may also be derived from a mammalian, and more particularly a human .
  • the catalytic domain may also comprise a barrel of seven pairs of helices ( ⁇ 7 ) , and the pairs of helices may consist of a first set of parallel hel ices inner-disposed in the barrel , and a second set of helices anti-parallel to the first set .
  • a method for determining a three- dimensional structure of an ⁇ l , 2 -mannosidase which comprises using a three-dimensional structure of ⁇ l , 2 - mannosidase of a yeast to derive a ⁇ l , 2 -mannosidase three-dimensional structure of another species therefrom.
  • the deriving may be effected by molecular replacement .
  • the three-dimensional structure may be from a mammal ian, and more particularly a human .
  • the f irst three-dimensional structure of the yeast ⁇ l , 2 -mannosidase may be a member of the class I ⁇ l , 2 -mannosidase family .
  • a method of using such a crystal in a drug screening assay comprises selecting a potent ial antagonist or inhibitor by performing rational drug design with the three-dimensional structure determined for the crystal , the selecting being performed in conj unction with computer modeling , adding the potential antagonist or inhibitor to a glycoprotein synthesis assay in which the ⁇ l , 2 - mannosidase is a rate-limiting factor, and detecting a change of protein synthesis , wherein a potent ial antagonist or inhibitor that inhibits maturation of carbohydrate on a newly formed glycoprotein and stabil izes a misfolded glycoprotein is selected as a potent ial drug .
  • Drugs may be screened for a specif ic inhibitor or antagonist of an ER ⁇ l , 2 -mannosidase enzyme , to stabil ize a misfolded glycoprotein in a genetic di sease .
  • the disease may consist of cystic f ibrosis or pulmonary emphysema
  • the glycoprotein may consist of a mutant of cystic f ibrosis transmembrane conductance regulator (CFTR) or ⁇ l - antitrypsin , respectively .
  • CFTR cystic f ibrosis transmembrane conductance regulator
  • This three-dimensional structure of the Class I processing ⁇ l , 2 -mannosidase of the present invention provides a framework to understand the mechanism of action, to determine the basis of the differences in specificity of the different family members and to elucidate their respective roles in glycoprotein maturation.
  • a expression vector comprising the nucleic acid of SEQ ID NO : 1 operatively associated with an expression control sequence
  • mutant forms of the ER ⁇ l,2- mannosidase enzyme have been determined to have altered specificity.
  • the knowledge of the structure of the active site of the yeast enzyme provided herein and the mode of substrate-binding may be used to develop specific inhibitors for preventing the degradation of abnormal, misfolded glycoproteins characteristic of genetic diseases including cystic fibrosis and emphysema.
  • active site cavity is intended to mean the active site within the barrel which is the region where the amino acid residues essential for catalysis and the essential calcium ion are located; i.e. it is where the action of the enzyme occurs during catalysis, where cleavage of mannose from the substrate occurs .
  • Fig. 1 illustrates the schematic ribbon representation of the three-dimensional structure of the yeast ⁇ l , 2 -mannosidase viewed down the ⁇ , barrel axis;
  • Fig. 2 illustrates the ribbon representation at 90° to the first orientation, and the protein-protein interaction in the crystal packing;
  • Fig. 3 illustrates a schematic representation of the high-mannose oligosaccharide HMI
  • Fig. 4 illustrates a detailed high-mannose oligosaccharide (H ) -enzyme interaction between HMI and the protein;
  • Fig. 5 illustrates a Van der Waals surface representation of the high-mannose oligosaccharide (HM) -enzyme interaction
  • Fig. 8A at the top illustrates the order of removal of mannose from Man 9GlcNAc 2 by 2 the R273L mutated form of the ER ⁇ l , 2 -mannosidase enzyme of the present invention .
  • a crystal of a class I ⁇ l , 2 -mannosidase a crystal of a class I ⁇ l , 2 -mannosidase .
  • the crystal structure of the catalytic domain, or active site, of the endoplasmic reticulum (ER) yeast ⁇ l , 2 -mannosidase that transforms Man 9 GlcNAc 2 to a single isomer of Man 8 GlcNAc 2 (isomer B lacking the ⁇ l,2-linked mannose on the ⁇ l,3 mannose of the ⁇ l , 6 branch) was determined at 1.5 Angstrom resolution.
  • the three-dimensional structure of this enzyme and of its active site may be used to design specific inhibitors that may stabilize misfolded glycoproteins in diseases.
  • the three-dimensional structure provided herein may also be used to determine the three-dimensional structure of other members of this enzyme family.
  • the catalytic domain of the yeast ⁇ l , 2 - mannosidase was produced in P. pastoris as a secreted glycoprotein (F. Lipari , A. Herscovics, Glycobiology 4, 697 (1994) ; F. Lipari, B. J. Gour-Salin, A. Herscovics, Bioche . Biophys. Res. Commun. 209, 322 (1995) ; F. Lipari and A. Herscovics, J. Biol. Chem. 271, 27615 (1996) ) .
  • Native and derivative data were first collected at room temperature using monochromated CuK ⁇ X- radiation (Rigaku Rotaflex RU200 rotating anode 0 generator) on a Mar Research (345 mm diameter) imaging plate system. Subsequent native data sets were collected on beamline X8C at the N.S.L.S. (Brookhaven National Laboratory, Upton, N-Y, U.S.A) using a Quantum4 CCD detector and flash-frozen crystals 5 (cryoprotected in artificial mother liquor containing 25% v/v glycerol) . All data were processed with DENZOTM and SCALEPACKTM.
  • the structure was determined using the single isomorphous replacement with anomalous scattering 0 (SIRAS) technique. This is the first processing enzyme in N-glycan biosynthesis whose three-dimensional structure has been determined. The structure has been refined to a R cr ⁇ st of 21.2% and a R £ree of 22.8% for the data between 50 and 1.54 A resolution, as may be seen 5 in Table 1. The structure of the yeast ER ⁇ l,2- mannosidase catalytic domain complexed with the inhibitor 1-deoxymannoj irimycin was also determined at 1.59 Angstroms resolution. This structure has been refined to a R cryst and R £ree of 21.6% and 24.4%, 0 respectively. Table 1
  • R cryst ⁇ I I F 0 I - I F c I I / ⁇ F 0 where F 0 and F c are the observed and calculated structure factors, respectively;
  • R sym ⁇ I I, - ⁇ I>
  • the final model contains residues 34 to 367, 371 to 409 and 411 to 549 as well as 414 solvent molecules, one glycerol molecule, one calcium ion and three N- glycans .
  • the ⁇ l , 2 - mannosidase catalytic domain is an ⁇ 7 helix barrel with overall dimensions of approximately 50 A x 50 A x 50 A.
  • This is the first example of an ⁇ -helix barrel consisting of seven pairs of helices. The molecule consists of consecutive helices alternating from outside to inside the barrel.
  • the LC side is structurally more complex and is similar to an "open flower” with strands forming the petals of the flower.
  • the ⁇ -strands pack together to form a series of anti-parallel ⁇ -sheets surrounding the helix-barrel.
  • the C-terminal of the protein (residues 512-549) consists of a ⁇ -hairpin protruding back into the center of the barrel from the SC side and an additional short helix, ⁇ l5.
  • the ⁇ -hairpin blocks the barrel, preventing the protein from looking like an open channel .
  • the ⁇ - hairpin, the inner helices and the ⁇ -sheets on the LC side of the barrel result in a cavity of approximately 15 A in depth, parallel to the central axis of the barrel, with a diameter of 25 A at the level of the ⁇ - sheets decreasing to approximately 10 A at the top of the ⁇ -hairpin.
  • This cavity is a consequence of the seven pairs of helices present in the barrel, as no significant cavity is found in ⁇ 6 -barrel proteins (A. Aleshin, A. Golubev, L. M. Firsov, R. B. Honzatko, J Biol. Chem. 267, 19291 (1992); P.M.
  • the ⁇ l , 2 linkage between mannose M7 and MIO of HMI is specifically cleaved by 5 the enzyme, yielding Man 8 GlcNAC 2 isomer B, the smallest oligosaccharide found in yeast N-glycans.
  • the residues as well as the glycerol molecule and the calcium ion are completely buried in the active site. E132 and D275, the putative catalytic residues,
  • the insert 10 are located at the entrance of a cavity.
  • the insert shows the position of the glycerol molecule, thought to mimic the oligosaccharide residue MIO.
  • residue M4 contacts R273 and R433, and M6 contacts R269, S272, D336, L338 and E399, whereas residue M5 of the ⁇ l, 3 -branch, contacts residues S184, S185, N129 and N196.
  • residue M7 that would form the target glycosidic bond with MIO in the substrate, is located towards the bottom of the catalytic site and binds to residues F131, E207, R273 and D275.
  • R273 may therefore together with R269, S272 and D336 stabilize the ⁇ l-6 arm of HMI and could potentially dictate the conformation of the oligosaccharide required by the enzyme to cleave the glycosidic linkage between M7 and MIO.
  • this structure can be used to determine the structure of the corresponding human ⁇ l , 2 -mannosidase.
  • molecular replacement can be employed, based on the yeast ER ⁇ l , 2 -mannosidase structure, to determine the human ⁇ l , 2 -mannosidase structure .
  • the specificity of the ER ⁇ l , 2 -mannosidase was changed by site-directed mutagenesis of a single amino acid residue that was seen to interact with the oligosaccharide in the crystal structure.
  • arginine 273 was replaced by leucine and the specificity of the ⁇ l , 2 -mannosidase produced in Pichia pastoris was altered.
  • the R273L mutant was found to remove additional mannose residues from Man 9 GlcNAc with Man S GlcNAc as the end product (Fig. 8A) .
  • Man 8 GlcNAc is formed by the parent enzyme (Fig. 8B) .
  • the oligosaccharides formed from [ 3 H] Man g GlcNAc were fractionated by HPLC, and the time course of oligosaccharide product formation was compared with that obtained for the parent enzyme (Fig.8B) .
  • R273L mutant is shown in Fig. 8A compared to the time course of trimming ⁇ ⁇ of Man 9 GlcNAc 2 to Man 8 GlcNAc 2 by 2 the wi ld type ER ⁇ l , 2 -mannosidase shown in Fig . 8B .
  • High resolution X H-NMR of the oligosaccharide intermediates formed by the R273L mutant showed that the order of mannose removal from Man 9 GlcNAc 2 , shown at the top c of
  • Fig. 8 is different from that previously observed for mammalian Golgi ⁇ l , 2 -mannosidases . Therefore, a single mutation in the catalytic domain of the yeast ER ⁇ l,2- annosidase produces an enzyme with novel specificity. Expression of the mutant ER ⁇ l , 2 -mannosidase intracellularly should modify the mannose trimming in the endoplasmic reticulum and may affect the fate of misfolded glycoproteins.
  • the yeast ⁇ l, 2 -mannosidase is an inverting glycosyl-hydrolase (F. Lipari, B. J. Gour-Salin, A.
  • the catalytic mechanism involves two acidic residues, one acting as a base removing a proton from water and the other acting as an acid donating a proton to the leaving group.
  • This hypothesis is supported by kinetic data showing a decrease in k cat , a change typical of glycosidases with mutations in their catalytic residues (F. Lipari and A.
  • the three-dimensional structure of the yeast ER ⁇ l , 2 -mannosidase complexed with the inhibitor 1- deoxymannoj irimycin was determined by X-ray crystallography.
  • the enzyme- inhibitor complex provides an understanding of the catalytic mechanism of the ⁇ l,2- mannosidase, the role of calcium and the role of the - 29 -
  • a single mutation in the catalytic domain of the yeast ER ⁇ l,2- mannosidase produces an enzyme with novel specificity.
  • Expression of the mutant ER ⁇ l,2-mannosidase mtracellularly should modify the mannose trimming in the endoplasmic reticulum and may affect the fate of misfolded glycoproteins.

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Abstract

Cristal et structure tridimensionnelle pour la famille enzymatique α1,2-mannosidase du réticulum endoplasmique, utiles pour l'identification de déterminants structuraux responsables de la spécificité des différentes enzymes de ladite famille et du développement d'inhibiteurs destinés à stabiliser les glycoprotéines anormales dans des maladies génétiques telles que la mucoviscidose et l'emphysème pulmonaire.
PCT/CA2000/001093 1999-09-23 2000-09-22 STRUCTURE TRIDIMENSIONNELLE ET CRISTAL D'UNE α1,2-MANNOSIDASE DE CLASSE I, ET PROCEDES D'UTILISATION DE CES SUBSTANCES WO2001021769A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003044185A3 (fr) * 2001-11-21 2004-08-05 Affinium Pharm Inc Nouveaux polypeptides purifies iimpliques dans un metabolisme general
FR2861991A1 (fr) * 2003-11-07 2005-05-13 Centre Nat Rech Scient Utilisation d'inhibiteurs de glucosidase pour une therapie de la mucoviscidose
TWI405850B (zh) * 2005-12-08 2013-08-21 Amgen Inc 經改良之宿主細胞及培養方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAMIRAND ET AL.: "Glycoprotein biosynthesis in Saccharomyces cerevisiae" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 23, 15 August 1991 (1991-08-15), pages 15120-15127, XP000993101 cited in the application *
DOLE ET AL.: "Crystallization and preliminary X-ray analysis of the class 1 aplha 1,2-mannosidase from Saccharomyces cerevisiae" JOURNAL OF STRUCTURAL BIOLOGY, vol. 120, no. 1, October 1997 (1997-10), pages 69-72, XP002166201 *
VALLÉE ET AL.: "Crystal structureof a class I alpha 1.2-mannosidase involved in N-glycan processing and endoplasmatic reticulum quality control" EMBO JOURNAL, vol. 19, no. 4, February 2000 (2000-02), pages 581-588, XP002166202 *
VALLÉE ET AL.: "Purification, crystallization and preliminary X-ray crystallographic analysis of recombinant murine Golgi mannosidase IA, a class I alpha-mannosidase involved in Asn-linked oligosaccharide maturation" ACTA CRYSTALLOGRAPHICA SECTION D BIOLOGICAL CRYSTALLOGRAPHY, vol. 55, no. 2, February 1999 (1999-02), pages 571-573, XP001001118 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003044185A3 (fr) * 2001-11-21 2004-08-05 Affinium Pharm Inc Nouveaux polypeptides purifies iimpliques dans un metabolisme general
FR2861991A1 (fr) * 2003-11-07 2005-05-13 Centre Nat Rech Scient Utilisation d'inhibiteurs de glucosidase pour une therapie de la mucoviscidose
WO2005046672A3 (fr) * 2003-11-07 2005-09-15 Centre Nat Rech Scient Utilisation d’inhibiteurs de glucosidase pour une therapie de la mucoviscidose
US7973054B2 (en) 2003-11-07 2011-07-05 Centre National De La Recherche Scientifique (C.N.R.S.) Use of glucosidase inhibitors for therapy of mucovisidosis
US8242136B2 (en) 2003-11-07 2012-08-14 Centre National De La Recherche Scientifique (C.N.R.S.) Use of glucosidase inhibitors for therapy of mucovisidosis
TWI405850B (zh) * 2005-12-08 2013-08-21 Amgen Inc 經改良之宿主細胞及培養方法

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