US2013266981A1PendingUtilityA1

Expression of class 2 mannosidase and class iii mannosidase in lower eukaryotic cells

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Assignee: GLYCOFI INCPriority: Feb 20, 2003Filed: Sep 21, 2012Published: Oct 10, 2013
Est. expiryFeb 20, 2023(expired)· nominal 20-yr term from priority
Inventors:Stephen Hamilton
C12P 21/005C12N 15/79C12N 9/1051C12Y 302/01114A01K 2217/075C12Y 302/01024C12N 9/2488
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Claims

Abstract

A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method for producing a human-like glycoprotein in a lower eukaryotic host cell comprising the step of expressing in the cell a mannosidase enzymatic activity that is capable of hydrolyzing an oligosaccharide substrate comprising either or both a Manα1,3 and Manα1,6 glycosidic linkage to the extent that at least 10% of the Manα1,3 and/or Manα1,6 linkages of the substrate are hydrolyzed in vivo. 
     
     
         2 . A method for producing a desired N-glycan in a lower eukaryotic host cell comprising the step of expressing in the cell a mannosidase enzymatic activity that is capable of hydrolyzing in vivo an oligosaccharide substrate comprising either or both a Manα1,3 and Manα1,6 glycosidic linkage wherein the desired N-glycan is produced within the host cell at a yield of at least 10 mole percent. The method of  claim 2 , wherein the desired N-glycan produced is selected from the group consisting of Man 3 GlcNAc 2 , GlcNAcMan 3 GlcNAc 2  and Man 4 GlcNAc 2 . 
     
     
         3 . The method of  claim 2 , wherein the desired N-glycan is characterized as having at least the oligosaccharide branch Manα1,3 (Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn. 
     
     
         4 . The method of  claim 1  or  2 , wherein the mannosidase enzymatic activity is capable of hydrolyzing in vivo both Manα1,3 and Manα1,6 linkages of an oligosaccharide substrate comprising a Manα1,3 and Manα1,6 glycosidic linkage. 
     
     
         5 . The method of  claim 1  or  2 , wherein the oligosaccharide substrate is characterized as Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,2 Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,2 Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn or high mannan. 
     
     
         6 . The method of  claim 1  or  2 , wherein the mannosidase activity is characterized as a Class 2 mannosidase activity. 
     
     
         7 . The method of  claim 6 , wherein the Class 2 mannosidase activity has a substrate specificity for GlcNAcβ1,2 Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; or GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn. 
     
     
         8 . The method of  claim 6 , wherein the Class 2 mannosidase activity is one which is normally found in the Golgi apparatus of a higher eukaryotic host cell. 
     
     
         9 . The method of  claim 1  or  2 , wherein the mannosidase activity is characterized as a Class IIx mannosidase activity. 
     
     
         10 . The method of  claim 9 , wherein the Class IIx mannosidase activity has a substrate specificity for Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; or Manα1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn. 
     
     
         11 . The method of  claim 1  or  2 , wherein the mannosidase activity is characterized as a Class III mannosidase activity. 
     
     
         12 . The method of  claim 11 , wherein the Class III mannosidase activity has a substrate specificity for (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; or high mannans. 
     
     
         13 . The method of  claim 1  or  2 , wherein the mannosidase activity is overexpressed. 
     
     
         14 . The method of  claim 1  or  2 , wherein the mannosidase is further capable of hydrolyzing a Manα1,2 linkage. 
     
     
         15 . The method of  claim 1  or  2 , wherein the mannosidase activity has a pH optimum of from about 5.0 to about 8.0. 
     
     
         16 . The method of  claim 1  or  2 , wherein the mannosidase is further capable of hydrolyzing a Manα1,2 linkage. 
     
     
         17 . The method of  claim 1  or  2 , wherein the mannosidase activity is localized within the secretory pathway of the host cell. 
     
     
         18 . The method of  claim 1  or  2 , wherein the mannosidase activity is expressed from a polypeptide localized within at least one of the ER, Golgi apparatus or the trans golgi network of the host cell. 
     
     
         19 . The method of  claim 1  or  2 , wherein the mannosidase activity is expressed from a nucleic acid encoding a polypeptide comprising a mannosidase catalytic domain fused to a cellular targeting signal peptide. 
     
     
         20 . The method of  claim 19 , wherein the mannosidase activity is expressed from a nucleic acid comprising sequences that encode a mannosidase catalytic domain native to the host cell 
     
     
         21 . The method of  claim 19 , wherein the mannosidase activity is expressed from a nucleic acid comprising sequences that encode a mannosidase catalytic domain heterologous to the host cell. 
     
     
         22 . The method of  claim 1  or  2 , wherein the mannosidase enzymatic activity is selected from the group consisting of  Arabidopsis thaliana  Mannosidase II,  C. elegans  Mannosidase II,  Ciona intestinalis  mannosidase II,  Drosophila  mannosidase II, Human mannosidase II, Mouse mannosidase II, Rat mannosidase II, Human mannosidase IIx, Insect cell mannosidase III, Human lysosomal mannosidase II and Human cytoplasmic mannosidase II. 
     
     
         23 . The method of  claim 1  or  2 , wherein the polypeptide is expressed from a nucleic acid comprising sequences that encode a target peptide native to the host cell. 
     
     
         24 . The method of  claim 1  or  2 , wherein the polypeptide is expressed from a nucleic acid comprising sequences that encode a target peptide heterologous to the mannosidase catalytic domain. 
     
     
         25 . The method of  claim 1  or  2 , further comprising the step of isolating the glycoprotein from the host cell. 
     
     
         26 . The method of  claim 1  or  2 , wherein the host cell is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.,  Fusarium gramineum, Fusarium venenatum  and  Neurospora crassa.    
     
     
         27 . The method of  claim 26 , wherein the host cell is  Pichia pastoris.    
     
     
         28 . The method of  claim 1  or  2 , wherein the glycoprotein is a therapeutic protein. 
     
     
         29 . The method of  claim 28 , wherein the therapeutic protein is selected from the group consisting of erythropoietin, cytokines, coagulation factors, soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins, urokinase, chymase, urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsin and α-feto protein. 
     
     
         30 . A nucleic acid library comprising at least two different genetic constructs, wherein at least one genetic construct comprises a nucleic acid fragment encoding a mannosidase class 2, IIx or III catalytic domain ligated in-frame with a nucleic acid fragment encoding a cellular targeting signal peptide which it is not normally associated with. 
     
     
         31 . The library of  claim 30 , wherein the mannosidase catalytic domain is selected from the group consisting of  Arabidopsis thaliana  Mannosidase II,  C. elegans  Mannosidase II,  Ciona intestinalis  mannosidase II,  Drosophila  mannosidase II, Human mannosidase II, Mouse mannosidase II, Rat mannosidase II, Human mannosidase IIx, Insect cell mannosidase III, Human lysosomal mannosidase II and Human cytoplasmic mannosidase II. 
     
     
         32 . The library of  claim 30 , wherein the nucleic acid fragment encoding a cellular targeting peptide is selected from the group consisting of:  Saccharomyces  GLS1,  Saccharomyces  MNS1,  Saccharomyces  SEC12,  Pichia  SEC,  Pichia  OCH1 , Saccharomyces  MNN9,  Saccharomyces  VAN1,  Saccharomyces  ANP1,  Saccharomyces  HOC1 , Saccharomyces  MNN10,  Saccharomyces  MNN11,  Saccharomyces  MNT1,  Pichia  D2,  Pichia  D9,  Pichia  J3,  Saccharomyces  KTR1,  Saccharomyces  KTR2,  Kluyveromyces  GnTI,  Saccharomyces  MNN2,  Saccharomyces  MNN5,  Saccharomyces  YUR1,  Saccharomyces  MNN1, and  Saccharomyces  MNN6. 
     
     
         33 . A vector comprising a fusion construct derived from a library of any one of  claims 30 - 32  operably linked to an expression control sequence, wherein said cellular targeting signal peptide is targeted to at least one of the ER, Golgi or trans-Golgi network. 
     
     
         34 . The vector of  claim 33 , wherein the expression control sequence is inducible or constitutive. 
     
     
         35 . The vector of  claim 33  which, upon expression in a host cell, encodes a mannosidase activity involved in producing GlcNAcMan 3 GlcNAc 2  Man 3 GlcNAc 2  or Man 4 GlcNAc 2  in vivo. 
     
     
         36 . A host cell comprising at least one vector of  claim 35 . 
     
     
         37 . A host cell comprising at least one vector selected from the group of vectors designated pKD53, pKD1, pKD5, pKD6 and pKD16. 
     
     
         38 . A chimeric polypeptide comprising a mannosidase catalytic domain fused in-frame to a targeting signal peptide and, upon expression in a lower eukaryotic host cell, capable of hydrolyzing in vivo an oligosaccharide substrate comprising either or both a Manα1,3 and Manα1,6 glycosidic linkage to the extent that at least 10% of the Manα1,3 and/or Manα1,6 linkages of the substrate are hydrolyzed in vivo. 
     
     
         39 . A chimeric polypeptide comprising a mannosidase catalytic domain fused in-frame to a targeting signal peptide and, upon expression in a lower eukaryotic host cell, capable of hydrolyzing in vivo an oligosaccharide substrate comprising a Manα1,3, Manα1,6, or Manα1,2 glycosidic linkage to the extent that a detectable moiety of the Manα1,3, Manα1,6 or Manα1,2 linkage of the substrate is hydrolyzed in vivo. 
     
     
         40 . A nucleic acid encoding a chimeric polypeptide of  claim 38 . 
     
     
         41 . A host cell comprising a chimeric polypeptide of  claim 38 . 
     
     
         42 . A host cell comprising a nucleic acid of  claim 40 . 
     
     
         43 . A glycoprotein produced in a host cell of  claim 41  or  claim 42 . 
     
     
         44 . An N-glycan produced in a host cell of  claim 41  or  claim 42 . 
     
     
         45 . The N-glycan of  claim 44 , wherein the N-glycan is characterized as uniform. 
     
     
         46 . A glycoprotein produced by the method of  claim 1  or  claim 2 . 
     
     
         47 . An N-glycan produced by the method of  claim 1  or  claim 2 . 
     
     
         48 . The N-glycan of  claim 47 , wherein the N-glycan is characterized as uniform. 
     
     
         49 . An isolated polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of:
 (a) SEQ ID NO: 92 ( C. elegans  FROM  FIG. 23 );   (b) at least about 90% similar to the amino acid residues of the donor nucleotide binding site of SEQ ID NO: 92;   (c) a nucleic acid sequence at least 92%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 93;   (d) a nucleic acid sequence that encodes a conserved polypeptide having the amino acid sequence of SEQ ID NO: 92;   (e) a nucleic acid sequence that encodes a polypeptide at least 78%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO:92;   (f) a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:92; and   (g) a nucleic acid sequence comprising a fragment of any one of (a)-(f) that is at least 60 contiguous nucleotides in length.   
     
     
         50 . An isolated polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of:
 (a) SEQ ID NO: 93(rat FROM  FIG. 23 );   (b) at least about 95% similar to the amino acid residues of the donor nucleotide binding site of SEQ ID NO: 93;   (c) a nucleic acid sequence at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 93;   (d) a nucleic acid sequence that encodes a conserved polypeptide having the amino acid sequence of SEQ ID NO: 93;   (e) a nucleic acid sequence that encodes a polypeptide at least 97%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 93;   (f) a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 93; and   (g) a nucleic acid sequence comprising a fragment of any one of (a)-(f) that is at least 60 contiguous nucleotides in length.   
     
     
         51 . An isolated polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of:
 (a) SEQ ID NO: 94(Ciona FROM  FIG. 23 );   (b) at least about 90% similar to the amino acid residues of the donor nucleotide binding site of SEQ ID NO: 94;   (c) a nucleic acid sequence at least 92%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 94;   (d) a nucleic acid sequence that encodes a conserved polypeptide having the amino acid sequence of SEQ ID NO: 94;   (e) a nucleic acid sequence that encodes a polypeptide at least 73%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 94;   (f) a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 94; and   (g) a nucleic acid sequence comprising a fragment of any one of (a)-(f) that is at least 60 contiguous nucleotides in length.   
     
     
         52 . An isolated polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of:
 (a) SEQ ID NO: 95(Arabidopsis FROM  FIG. 23 );   (b) at least about 95% similar to the amino acid residues of the donor nucleotide binding site of SEQ ID NO: 95;   (c) a nucleic acid sequence at least 96%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 95;   (d) a nucleic acid sequence that encodes a conserved polypeptide having the amino acid sequence of SEQ ID NO: 95;   (e) a nucleic acid sequence that encodes a polypeptide at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 95;   (f) a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 95; and   (g) a nucleic acid sequence comprising a fragment of any one of (a)-(f) that is at least 60 contiguous nucleotides in length.   
     
     
         53 . A modified polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of the conserved regions SEQ ID NO: 5-SEQ ID NO: 15 wherein the encoded polypeptide is involved in hydrolyzing a Manα1,3 and/or a Manα1,6 glycosidic linkage of an oligosaccharide. 
     
     
         54 . A modified polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of the conserved regions of SEQ ID NO: 49-SEQ ID NO: 59 wherein the encoded polypeptide is involved in hydrolyzing a Manα1,3 and/or a Manα1,6 glycosidic linkage of an oligosaccharide. 
     
     
         55 . A vector selected from the group consisting of pKD53, pKD1, pKD5, pKD6 and pKD16.

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