WO1999002706A1 - Peptides de glycosylation - Google Patents
Peptides de glycosylation Download PDFInfo
- Publication number
- WO1999002706A1 WO1999002706A1 PCT/GB1998/001989 GB9801989W WO9902706A1 WO 1999002706 A1 WO1999002706 A1 WO 1999002706A1 GB 9801989 W GB9801989 W GB 9801989W WO 9902706 A1 WO9902706 A1 WO 9902706A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequence
- recombinant dna
- seq
- mpt83
- glycosylation
- Prior art date
Links
- 230000013595 glycosylation Effects 0.000 title claims abstract description 32
- 238000006206 glycosylation reaction Methods 0.000 title claims abstract description 31
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 29
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 21
- 101150023079 mpt83 gene Proteins 0.000 claims abstract description 35
- 229920001184 polypeptide Polymers 0.000 claims abstract description 19
- 108020004511 Recombinant DNA Proteins 0.000 claims abstract description 16
- 229960005486 vaccine Drugs 0.000 claims abstract description 11
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims abstract description 10
- 239000012634 fragment Substances 0.000 claims abstract description 10
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims abstract description 8
- 108020004414 DNA Proteins 0.000 claims abstract description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229960001230 asparagine Drugs 0.000 claims abstract description 5
- 235000009582 asparagine Nutrition 0.000 claims abstract description 5
- 241001446247 uncultured actinomycete Species 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 5
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000028993 immune response Effects 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 230000001681 protective effect Effects 0.000 claims description 2
- 230000000405 serological effect Effects 0.000 claims description 2
- 241000124008 Mammalia Species 0.000 claims 3
- 102000053602 DNA Human genes 0.000 claims 1
- 238000002649 immunization Methods 0.000 claims 1
- 230000000069 prophylactic effect Effects 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 11
- 102000004169 proteins and genes Human genes 0.000 abstract description 10
- 235000018102 proteins Nutrition 0.000 abstract description 9
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 241000186361 Actinobacteria <class> Species 0.000 abstract 1
- 230000002163 immunogen Effects 0.000 abstract 1
- 150000001413 amino acids Chemical group 0.000 description 19
- 101150056832 mpt70 gene Proteins 0.000 description 14
- 235000001014 amino acid Nutrition 0.000 description 13
- 229940024606 amino acid Drugs 0.000 description 13
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 201000008827 tuberculosis Diseases 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 241000187480 Mycobacterium smegmatis Species 0.000 description 4
- 230000004988 N-glycosylation Effects 0.000 description 4
- 230000004989 O-glycosylation Effects 0.000 description 4
- 238000003119 immunoblot Methods 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 241000186366 Mycobacterium bovis Species 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 108010062580 Concanavalin A Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 101001057129 Bacillus cereus Enterotoxin Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000282346 Meles meles Species 0.000 description 1
- 206010062207 Mycobacterial infection Diseases 0.000 description 1
- 101100131080 Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97) mpb70 gene Proteins 0.000 description 1
- 101000700655 Mycobacterium leprae (strain TN) Serine-rich antigen Proteins 0.000 description 1
- 101001043272 Mycobacterium tuberculosis (strain ATCC 25177 / H37Ra) Lipoprotein LpqH Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 125000000625 hexosyl group Chemical group 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 101150006328 mpb83 gene Proteins 0.000 description 1
- 208000027531 mycobacterial infectious disease Diseases 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- the present invention relates to novel polypeptides which include glycosylation motifs, to DNA encoding said polypeptides, and to the use of these moieties for example in vaccines.
- WO97/083222 describes and claims a gene mpt83 which encodes the glycosylated protein MPT83 of M. tuberculosis . It has previously been reported that MPT83 is post-translationally modified by the fast growing mycobacterium M. smegmatis (R.G. Hewinson et al., Scand. J. Immunol. 43 (1996) pp 490-499).
- glycosylation motif a smaller motif will act as a glycosylation motif.
- a further glycosylation site within the PT83 protein has been identified. Such sites may advantageously be introduced into other proteins particularly when these are intended to be used as vaccines . Glycosylation may improve the immunogenicity of the protein. Furthermore, it has been found that in some instances, glycosylation leads to improved stability of the protein, leading for example to a longer and stronger immune response.
- the invention provides a recombinant DNA which encodes a polypeptide which is other than a wild-type or native polypeptide and which is glycosylated when expressed in an actinomycete host, said DNA comprising a sequence which encodes the amino acid sequence
- PAAPVTTAA SEQ ID NO 1
- fragment or variant thereof including at least both the threonine residues and being able to act as a glycosylation motif;
- the term " fragment” includes small peptide units for example of 5 amino acids or more.
- variant refers to peptide units wherein one or more amino acids, for example up to 4 amino acids, have been changed whilst not altering the qualitative function of the peptide.
- the changes may be what are regarded as " conservative substitutions" where amino acids are replaced by amino acids of an essentially similar nature (e.g. basicity etc.) in which case rather more subtitutions may be tolerated.
- Alternatively a small number of non-conservative substitutions may be introduced without altering the function of the peptide, as would be determinable by the skilled person.
- the recombinant DNA of the invention comprises a sequence encoding SEQ ID No 1 as defined above .
- a sequence may be the sequence found in the mpt83 gene, i.e.
- a futher glycosylation motif of MPT83 involves the asparagine at position 192 in the sequence. It is probable that this residue forms part of a glycosylation motif located between 179-196 of MPT83.
- the motif is of at least 5 amino acids, and preferably at least 10 amino acids in length. The entire 20 amino acid sequence is of sequence
- recombinant DNA of the invention may encode said sequence (SEQ ID No. 3) .
- the DNA may comprise the sequence as found in mpt83 , i.e.
- Polypeptides obtainable by expression of a recombinant DNA as described above form a further aspect of the invention.
- Such polypeptides may be obtained by incorporating recombinant DNA as described above into a replication vector, transforming a suitable host cell with said vector and culturing the cell.
- Methods of preparing polypeptides as well as vectors and transformed cells used in the method form further aspects of the invention.
- Suitable host cells would be determinable by the skilled person using routine methods. However, preferably the host cell is an actinomycete such as a mycobacterial cell.
- modified polypeptides of the invention and especially the DNA encoding said polypeptides are explained in WO97/083222.
- these modified polypeptides would be useful in vaccines, especially those protective against mycobacterial infection such a tuberculosis, since they may improve immunogenicity and stability as discussed above.
- they would provide a serological marker allowing the vaccine strain to be distinguished from infection by a native strain.
- Vaccines may be in the form of live or " killed” vaccines as is understood in the art.
- the " naked DNA” approach to vaccines would be amenable to the use of the present invention.
- Vaccines may be in the form of pharmaceutical compositions in combination with a pharmaceutically acceptable carrier.
- FIG. 1 illustrates the E. coli alkaline phosphatse (PhoA) reporter system
- Figure 2 shows the alignment of amino acid sequences of MPT83 and MPT70;
- Figure 3 illustrates the modification of MPT70 to introduce a glycosylation site
- Figure 4 shows a range of constructs tested for recognition by ConA
- Figure 5 illustrates a proposed model for glycosylation of the antigen MPT83.
- Sonicated extracts of recombinant M. smegmatis expressing the above constructs were tested for ConA binding after fractionation by SDS-PAGE and transfer to nitrocellulose membranes. Levels of protein expression were determined by anti-PhoA immunoblotting. Equal expression of the MPT70 and MPT83-PhoA fusions were confirmed by the immunoblot ing with anti-PhoA. The MPT83-PhoA fusion was recognised by ConA whereas the MPT70-PhoA fusion was not. Therefore, it was concluded that the signal motif for glycosylation of MPT83 lies within the amino acid residues that differ from those of MPT70 (See Figure 2) .
- PAAPVTTAA amino acid sequence (SEQ ID No 1) (residues 43-51) that was thought to be similar to the sequence of a glycopeptide, PAPPVPTTA, from a 45kda protein of M. tuberculosis (K.M. Dobos et al., Infect Immun 63 (1995) 2846-53). This latter peptide was shown to be ) -glycosidically linked to a disaccharide composed of two hexose residues through a threonine residue.
- Example 3 Immunoblotting was effected as described in Example 2. This showed that mutation of the threonine residues ablated ConA binding. There was also a noteable decrease in the apparent molecular weight of the mutated fusion protein. This result suggests that the peptide region of SEQ Id No. 1 is a signal motif for O-linked glycosylation in MPT83.
- Example 4 my ⁇ osylation of MPT70 using the signal motif of MPT83 The corresponding regions of the MPT83 and MPT70 proteins is shown in Table 3.
- N-linked glycosylation site within MPT83 Further comparison of the amino acid sequence of MPT83 and MPT70 identified manner differences in amino acid sequence that might generate motifs for both 0 and N-linked glycosylation in MPT83.
- a putative site for N-linked glycosylation was identifed within the carboxy terminus of MPT83 at asparagine residue 193. This residue was mutated to glycine (the corresponding residue in MPT70) in both the native molecule and a recombinant MPT83 lacking the O-glycosylation site at threonine residues 48 and 49 as shown in Figure 4.
- the recognition of the constructs by ConA is also shown in Figure 4.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
L'invention concerne un ADN recombinant qui code un polypeptide (autre qu'un polypeptide de type sauvage ou natif) glycosylé lorsqu'il est exprimé dans un hôte actinomycète. Cet ADN comprend une séquence qui code la séquence d'acide aminé PAAPVTTAA (SEQ ID N° 1), ou un fragment ou variant de ladite séquence incluant au moins les résidus thréonines et pouvant agir comme motif de glycosylation, et/ou une séquence codant un motif de glycosylation trouvé dans la région de la protéine MPT83 contenant une asparagine à la position 192 dans la séquence DLTVIGARDDLMVNNAGL (SEQ ID N° 3) ou un fragment de ladite séquence. Ces ADN recombinants conviennent pour la production de vaccins étant donné leur aptitude à fournir un site de glycosylation, reconnu en particulier par des actinomycètes. La glycosylation est préférée en ce qu'elle peut améliorer l'immunogénéicité et la stabilité d'une protéine immunogène.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU82319/98A AU8231998A (en) | 1997-07-07 | 1998-07-06 | Glycosylation peptides |
| GB9930098A GB2341606A (en) | 1997-07-07 | 1998-07-06 | Glycosylation peptides |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9714242.6A GB9714242D0 (en) | 1997-07-07 | 1997-07-07 | Glycosylation peptides |
| GB9714242.6 | 1997-07-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1999002706A1 true WO1999002706A1 (fr) | 1999-01-21 |
Family
ID=10815445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/GB1998/001989 WO1999002706A1 (fr) | 1997-07-07 | 1998-07-06 | Peptides de glycosylation |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU8231998A (fr) |
| GB (2) | GB9714242D0 (fr) |
| WO (1) | WO1999002706A1 (fr) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992014823A1 (fr) * | 1991-02-14 | 1992-09-03 | N.V. Innogenetics S.A. | Polypeptides de mycobacterium et acides nucleiques les codant utilises dans le diagnostic et le traitement de la tuberculose |
| WO1994005780A1 (fr) * | 1992-08-28 | 1994-03-17 | The Public Health Research Institute Of The City Of New York, Inc. | Glycoproteines de fusion |
| WO1997008322A1 (fr) * | 1995-08-25 | 1997-03-06 | The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Gene mpt83 extrait de mycobacterium tuberculosis |
-
1997
- 1997-07-07 GB GBGB9714242.6A patent/GB9714242D0/en not_active Ceased
-
1998
- 1998-07-06 GB GB9930098A patent/GB2341606A/en not_active Withdrawn
- 1998-07-06 AU AU82319/98A patent/AU8231998A/en not_active Abandoned
- 1998-07-06 WO PCT/GB1998/001989 patent/WO1999002706A1/fr active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992014823A1 (fr) * | 1991-02-14 | 1992-09-03 | N.V. Innogenetics S.A. | Polypeptides de mycobacterium et acides nucleiques les codant utilises dans le diagnostic et le traitement de la tuberculose |
| WO1994005780A1 (fr) * | 1992-08-28 | 1994-03-17 | The Public Health Research Institute Of The City Of New York, Inc. | Glycoproteines de fusion |
| WO1997008322A1 (fr) * | 1995-08-25 | 1997-03-06 | The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Gene mpt83 extrait de mycobacterium tuberculosis |
Non-Patent Citations (1)
| Title |
|---|
| POWELL M F ET AL: "PEPTIDE STABILITY IN DRUG DEVELOPMENT. II EFFECT OF SINGLE AMINO ACID SUBSTITUTION AND GLYCOSYLATION ON PEPTIDE REACTIVITY IN HUMAN SERUM", PHARMACEUTICAL RESEARCH, vol. 10, no. 9, 1 September 1993 (1993-09-01), pages 1268 - 1273, XP000569558 * |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2341606A (en) | 2000-03-22 |
| GB9930098D0 (en) | 2000-02-09 |
| AU8231998A (en) | 1999-02-08 |
| GB9714242D0 (en) | 1997-09-10 |
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