Human glycine transporter type 2

• US 20030165907A1
• Filed: 07/09/2002
• Published: 09/04/2003
• Est. Priority Date: 11/12/1998
• Status: Abandoned Application

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Gliatech Inc.
• Inventors: Burgess, Loyd H., Gallagher, Michael J., Brunden, Kurt R.
• Application Number:
  US10/191,813
• Publication Number:
  US 20030165907A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: C07K 14/47 from mammals

Abstract

Nucleic acids and proteins derived from sequences of the human glycine transporter type 2 are described.

Claims

1. A purified nucleic acid comprising a nucleic acid sequence that encodes a glycine transporter type 2 (GlyT2) amino acid sequence that has glycine transport activity and that has a serine at a position corresponding to amino acid 24 of SEQ ID NO:

124.

9. A purified nucleic acid according to any one of claims 1-8, wherein the encoded GlyT2 amino acid sequence is that of amino acids 1 to 797 of SEQ ID NO:

124 but having one or more of the following amino acid substitutions;

(1) serine at position 24, (2) tryptophan at position 74, (3) glycine at position 155, (4) aspartic acid at position 188, (5) leucine at position 362, (6) alanine at a position corresponding to amino acid 431, or (7) serine at position 582.

10. A purified nucleic acid according to claim 9, wherein the encoded GlyT2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:

122.

11. A purified nucleic acid according to claim 9, wherein the encoded GlyT2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:

120.

12. A purified nucleic acid according to any one of claims 1-9, wherein the nucleic acid sequence is that of bases 1 to 2391 of SEQ ID NO:

123 but having one or more of the following nucleotide substitutions;

(1) A at position 70, (2) T at position 220, (3) G at position 463, (4) G at position 562, (5) T at position 1085, (6) C at position 1292, (7) A at position 1299, (8) T at position 1617, or (9) T at position 1744.

13. A purified nucleic acid according to claim 12, wherein the nucleic acid sequence is that of SEQ ID NO:

121.

14. A purified nucleic acid according to claim 12, wherein the nucleic acid sequence is that of SEQ ID NO:

119.

15. A purified nucleic acid according to any one of claims 1-9, that additionally has one or more of the following amino acids:

(1) leucine at a position corresponding to amino acid 26, (2) leucine at a position corresponding to amino acid 75, (3) valine at a position corresponding to amino acid 89, (4) glycine at a position corresponding to amino acid 102, (5) glutamic acid at a position corresponding to amino acid 174, (6) proline at a position corresponding to amino acid 195, (7) glycine at a position corresponding to amino acid 199, (8) leucine at a position corresponding to amino acid 249, (9) proline at a position corresponding to amino acid 306, (10) glutamic acid at a position corresponding to amino acid 419, (11) asparagine at a position corresponding to amino acid 442, (12) lysine at a position corresponding to amino acid 455, (13) cysteine at a position corresponding to amino acid 458, (14) proline at a position corresponding to amino acid 485, (15) arginine at a position corresponding to amino acid 493, or (16) glutamic acid at a position corresponding to amino acid 650, of SEQ ID NO;

124.

16. A purified nucleic acid according to any one of claims 1-9, that additionally has one or more of the following amino acids:

(1) phenylalanine at a position corresponding to amino acid 124, (2) asparagine at a position corresponding to amino acid 279, (3) glycine at a position corresponding to amino acid 393, (4) asparagine at a position corresponding to amino acid 457, (5) asparagine at a position corresponding to amino acid 463, (6) tyrosine at a position corresponding to amino acid 610, (7) valine at a position corresponding to amino acid 611, (8) serine at a position corresponding to amino acid 733, (9) valine at a position corresponding to amino acid 735, (10) leucine at a position corresponding to amino acid 245, (11) leucine at a position corresponding to amino acid 305, (12) isoleucine at a position corresponding to amino acid 366, or (13) proline at a position corresponding to amino acid 400, of SEQ ID NO;

124.

17. A purified nucleic acid according to claim 12, but additionally having has one or more of the following nucleotide substitutions:

(a) T at position 77, (b) T at position 220, (c) T at position 244, (d) T at position 266, (e) G at position 304, (f) A at position 521, (g) C at position 583, (h) G at position 596, (i) G at position 678, (j) C at position 681, (k) T at position 745, (l) C at position 750, (m) T at position 765, (n) C or A at position 777, (o) G at position 867, (p) C at position 917, (q) A at position 1256, (r) C at position 1292, (s) A at position 1325, (t) A at position 1364, (u) C at position 1374, (v) A at position 1392, (w) C at position 1454, (x) G at position 1478, (y) C at position 1854, (z) A at position 1949, (aa) C at position 1959, or (bb) C at position 2130.

18. A purified nucleic acid according to claim 12, but additionally having one or more of the following nucleotide substitutions:

(a) C at position 6, (b) T at position 371, (c) T at position 571, (d) A at position 836, (e) G at position 1116, (f) G at position 1177, (g) C at position 1371, (h) A at position 1387, (i) A at position 1829, (j) G at position 1831, (k) C at position 2198, (l) G at position 2203, (m) G at position 342, (n) C at position 733, (o) C at position 913, (p) A at position 951, (q) T at position 1097, (r) C at position 1199, (s) T at position 352, or (t) A at position 2103.

19. A vector comprising the nucleic acid sequence of any one of claims 1-8, 10-11, and 13-14.

20. A vector comprising the nucleic acid sequence of claim 9.

21. A vector comprising the nucleic acid sequence of claim 12.

22. A vector comprising the nucleic acid sequence of claim 15.

23. A vector comprising the nucleic acid sequence of claim 16.

24. A vector comprising the nucleic acid sequence of claim 17.

25. A vector comprising the nucleic acid sequence of claim 18.

26. A recombinant host cell containing the vector of claim 19.

27. A recombinant host cell containing the vector of claim 20.

28. A recombinant host cell containing the vector of claim 21.

29. A recombinant host cell containing the vector of claim 22.

30. A recombinant host cell containing the vector of claim 23.

31. A recombinant host cell containing the vector of claim 24.

32. A recombinant host cell containing the vector of claim 25.

33. A recombinant host cell containing the nucleic acid sequence of any one of claims 1-8, 10-11, and 13-14.

34. A recombinant host cell containing the nucleic acid sequence of claim 9.

35. A recombinant host cell containing the nucleic acid sequence of claim 12.

36. A recombinant host cell containing the nucleic acid sequence of claim 15.

37. A recombinant host cell containing the nucleic acid sequence of claim 16.

38. A recombinant host cell containing the nucleic acid sequence of claim 17.

39. A recombinant host cell containing the nucleic acid sequence of claim 18.

40. A method of making GlyT2 comprising expressing the GlyT2 in the recombinant host cell of claim 33.

41. A method of making GlyT2 comprising expressing the GlyT2 in the recombinant host cell of claim 34.

42. A method of making GlyT2 comprising expressing the GlyT2 in the recombinant host cell of claim 35.

43. A method of making GlyT2 comprising expressing the GlyT2 in the recombinant host cell of claim 36.

44. A method of making GlyT2 comprising expressing the GlyT2 in the recombinant host cell of claim 37.

45. A method of making GlyT2 comprising expressing the GlyT2 in the recombinant host cell of claim 38.

46. A method of making GlyT2 comprising expressing the GlyT2 in the recombinant host cell of claim 39.

2. A purified nucleic acid comprising a nucleic acid sequence that encodes a GlyT2 amino acid sequence that has glycine transport activity and that has a tryptophan at a position corresponding to amino acid 74 of SEQ ID NO:

124.

3. A purified nucleic acid comprising a nucleic acid sequence that encodes a GlyT2 amino acid sequence that has glycine transport activity and that has a glycine at a position corresponding to amino acid 155 of SEQ ID NO:

124.

4. A purified nucleic acid comprising a nucleic acid sequence that encodes a GlyT2 amino acid sequence that has glycine transport activity and that has an aspartic acid at a position corresponding to amino acid 188 of SEQ ID NO:

124.

5. A purified nucleic acid comprising a nucleic acid sequence that encodes a GlyT2 amino acid sequence that has glycine transport activity and that has a leucine at a position corresponding to amino acid 362 of SEQ ID NO:

124.

6. A purified nucleic acid comprising a nucleic acid sequence that encodes a GlyT2 amino acid sequence that has glycine transport activity and that has an alanine at a position corresponding to amino acid 431 of SEQ ID NO:

124.

7. A purified nucleic acid comprising a nucleic acid sequence that encodes a GlyT2 amino acid sequence that has glycine transport activity and that has a serine at a position corresponding to amino acid 582 of SEQ ID NO:

124.

8. A purified nucleic acid comprising a nucleic acid sequence that encodes a GlyT2 amino acid sequence that has glycine transport activity and that has one or more of the following amino acids:

(1) serine at a position corresponding to amino acid 24, (2) tryptophan at a position corresponding to amino acid 74, (3) glycine at a position corresponding to amino acid 155, (4) aspartic acid at a position corresponding to amino acid 188, (5) leucine at a position corresponding to amino acid 362, (6) alanine at a position corresponding to amino acid 431, or (7) serine at a position corresponding to amino acid 582, of SEQ ID NO;

124.

47. A purified nucleic acid encoding a glycine transporter type 2 (GlyT2) having at least about 96% sequence identity with a reference sequence which is the protein sequence of SEQ ID NO:

122 or with a sequence corresponding to the protein sequence of SEQ ID NO;

122 except that it has one or more of the following amino acid substitutions;

(1) Pro²⁶ to Leu, (2) Arg⁷⁴ to Trp, (3) Pro⁷⁵ to Leu, (4) Ala⁸⁹ to Val, (5) Ser¹⁰² to Gly, (6) Val¹⁷⁴ to Glu, (7) Ser¹⁹⁵ to Pro, (8) Asp¹⁹⁹ to Gly, (9) Val²⁴⁹ to Leu, (10) Leu³⁰⁶ to Pro, (11) Gly⁴¹⁹ to Glu, (12) Thr⁴⁴² to Asn, (13) Thr⁴⁵⁵ to Lys, (14) Trp⁴⁵⁸ to Cys, (15) Leu⁴⁸⁵ to Pro, (16) Lys⁴⁹³ to Arg, or (17) Val⁶⁵⁰ to Glu.

48. The nucleic acid of claim 47, wherein the reference sequence is the protein sequence of SEQ ID NO:

122 or with a sequence corresponding to the protein sequence of SEQ ID NO;

122 except that it has one or more of the following amino acid substitutions;

(1) Pro²⁶ to Leu, (2) Arg⁷⁴ to Trp, (3) Pro⁷⁵ to Leu, (4) Ala⁸⁹ to Val, (5) Ser¹⁰² to Gly, (6) Val¹⁷⁴ to Glu, (7) Ser¹⁹⁵ to Pro, (8) Asp¹⁹⁹ to Gly, (9) Val²⁴⁹ to Leu, (10) Leu³⁰⁶ to Pro, (11) Gly⁴¹⁹ to Glu, (12) Thr⁴⁴² to Asn, (13) Thr⁴⁵⁵ to Lys, (14) Trp⁴⁵⁸ to Cys, (15) Leu⁴⁸⁵ to Pro, (16) Lys⁴⁹³ to Arg, or (17) Val⁶⁵⁰ to Glu.

49. The nucleic acid of claim 47, wherein the sequence identity is selected from the group of at least about 97%, at least about 98% and at least about 99% sequence identity.

50. The nucleic acid of claim 47, wherein the sequence identity is at least about 99.5%.

51. The nucleic acid of claim 47, wherein the nucleic acid encodes a GlyT2 having the reference sequence.

52. The nucleic acid of claim 47, comprising the nucleic acid sequence of SEQ ID NO:

121 or with a sequence that varies from the nucleic acid sequence of SEQ ID NO;

121 by having one or more of the following nucleotide substitutions;

(a) C⁷⁷→

T, (b) C²²⁰→

T, (c) C²²⁴→

T, (d) C²⁶⁶→

T, (e) A³⁰⁴→

G, (f) T⁵²¹→

A, (g) T⁵⁸³→

C, (h) A⁵⁹⁶→

G, (i) A⁶⁷⁸→

G, (j) T⁶⁸¹→

C, (k) G⁷⁴⁵→

T, (l) G⁷⁵⁰→

C, (m) C⁷⁶⁵→

T, (n) G⁷⁷⁷→

C, (o) A⁸⁶⁷→

G, (p) T⁹¹⁷→

C, (q) G¹²⁵⁶→

A, (r) T¹²⁹²→

C, (s) C¹³²⁵→

A, (t) C¹³⁶⁴→

A, (u) G¹³⁷⁴→

C, (v) C¹³⁹²→

A, (w) T¹⁴⁵⁴→

C, (x) A¹⁴⁷⁸→

G, (y) T¹⁸⁵⁴→

C, (z) T¹⁹⁴⁹→

A, (aa) T¹⁹⁵⁹→

C, or (bb) T²¹³⁰→

C.

54. A vector comprising the nucleic acid of claim 47 and an extrinsic promoter functionally associated therewith.

57. A cell containing the nucleic acid of any one of claims 47-53 and 55-56.

58. A cell transformed with a vector according to claim 54.

59. A method of producing a glycine transporter comprising growing the cells of claim 57.

60. A method of producing a glycine transporter comprising growing the cells of claim 58.

61. The method of claim 59 further comprising at least one of (a) isolating membranes from the cells, which membranes comprise the glycine transporter or (b) extracting a protein fraction from the cells which fraction comprises the glycine transporter.

62. The method of claim 60 further comprising at least one of (a) isolating membranes from the cells, which membranes comprise the glycine transporter or (b) extracting a protein fraction from the cells which fraction comprises the glycine transporter.

63. A glycine transporter isolated from a cell according to claim 57.

64. A glycine transporter isolated from a cell according to claim 58.

65. A method for characterizing a bioactive agent for treatment of a nervous system disorder or condition or for identifying bioactive agents for treatment of a nervous system disorder or condition, the method comprising (a) providing a first assay composition comprising (i) a cell according to claim 57 or (ii) an isolated glycine transporter protein according to claim 63, (b) contacting the first assay composition with the bioactive agent or a prospective bioactive agent and measuring the amount of glycine transport exhibited by the assay composition.

66. A method for characterizing a bioactive agent for treatment of a nervous system disorder or condition or for identifying bioactive agents for treatment of a nervous system disorder or condition, the method comprising (a) providing a first assay composition comprising (i) a cell according to claim 58 or (ii) an isolated glycine transporter protein (ii) an isolated glycine transporter protein according to claim 64, (b) contacting the first assay composition with the bioactive agent or a prospective bioactive agent and measuring the amount of glycine transport exhibited by the assay composition.

67. The method of claim 65, further comprising comparing the amount of glycine transport exhibited by the first assay composition with the amount of glycine transport exhibited by a second such assay composition that is treated the same as the first assay composition except that it is not contacted with the bioactive agent or prospective bioactive agent.

68. The method of claim 66, further comprising comparing the amount of glycine transport exhibited by the first assay composition with the amount of glycine transport exhibited by a second such assay composition that is treated the same as the first assay composition except that it is not contacted with the bioactive agent or prospective bioactive agent.

69. The method of claim 65, wherein the nervous system disorder or condition is one of the group consisting of (a) pain, (b) spasticity, (c) myoclonus, (d) muscle spasm, (e) muscle hyperactivity or (f) epilepsy.

70. The method of claim 66, wherein the nervous system disorder or condition is one of the group consisting of (a) pain, (b) spasticity, (c) myoclonus, (d) muscle spasm, (e) muscle hyperactivity or (f) epilepsy.

71. The method of claim 69, wherein the spasticity is associated with stroke, head trauma, neuronal cell death, multiple sclerosis, spinal cord injury, dystonia, Huntington'"'"'s disease or amyotrophic lateral sclerosis.

72. The method of claim 70, wherein the spasticity is associated with stroke, head trauma, neuronal cell death, multiple sclerosis, spinal cord injury, dystonia, Huntington'"'"'s disease or amyotrophic lateral sclerosis.

81. A method for preparing an antibody specific for human GlyT2 comprising immunizing an animal with an isolated glycine transporter protein according to claim 63.

82. A method for preparing an antibody specific for human GlyT2 comprising immunizing an animal with an isolated glycine transporter protein according to claim 64.

53. The nucleic acid sequence of SEQ ID NO:

121 or a sequence that varies from the nucleic acid sequence of SEQ ID NO;

121 by having one or more of the following nucleotide substitutions;

C²²⁰→

T, A³⁰⁴→

G, or T¹²⁹²→

C.

55. A nucleic acid encoding a glycine transporter protein having at least about 99.5% sequence identity with all or one to two contiguous portions of a reference amino acid sequence, wherein the reference sequence is SEQ ID NO:

122 or an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO;

122 except that it has one or more of the following amino acid substitutions;

(1) Pro²⁶ to Leu, (2) Arg⁷⁴ to Trp, (3) Pro⁷⁵ to Leu, (4) Ala⁸⁹ to Val, (5) Ser¹⁰² to Gly, (6) Val¹⁷⁴ to Glu, (7) Ser¹⁹⁵ to Pro, (8) Asp¹⁹⁹ to Gly, (9) Val²⁴⁹ to Leu, (10) Leu³⁰⁶ to Pro, (11) Gly⁴¹⁹ to Glu, (12) Thr⁴⁴² to Asn, (13) Thr⁴⁵⁵ to Lys, (14) Trp⁴⁵⁸ to Cys, (15) Leu⁴⁸⁵ to Pro, (16) Lys⁴⁹³ to Arg, or (17) Val⁶⁵⁰ to Glu.

56. The nucleic acid of claim 55, wherein the one to two contiguous sequence portions comprise amino acids selected from the group of at least about 600 amino acids, at least about 700 amino acids and at least about 750 amino acids.

73. A nucleic acid that hybridizes with a reference nucleic acid sequence which is SEQ ID NO:

121 or with a sequence that varies from the nucleic acid sequence of SEQ ID NO;

121 by having one or more of the following nucleotide substitutions;

(a) C⁷⁷→

T, (b) C²²⁰→

T, (c) C²²⁴→

T, (d) C²⁶⁶→

T, (e) A³⁰⁴→

G, (f) T⁵²¹→

A, (g) T⁵⁸³→

C, (h) A⁵⁹⁶→

G, (i) A⁶⁷⁸→

G, (j) T⁶⁸¹→

C, (k) G⁷⁴⁵→

T, (l) G⁷⁵⁰→

C, (m) C⁷⁶⁵→

T, (n) G⁷⁷⁷→

C, (o) A⁸⁶⁷→

G, (p) T⁹¹⁷→

C, (q) G¹²⁵⁶→

A, (r) T¹²⁹²→

C, (s) C¹³²⁵→

A, (t) C¹³⁶⁴→

A, (u) G¹³⁷⁴→

C, (v) C¹³⁹²→

A, (w) T¹⁴⁵⁴→

C, (x) A¹⁴⁷⁸→

G, (y) T¹⁸⁵⁴→

C, (z) T¹⁹⁴⁹→

A, (aa) T¹⁹⁵⁹→

C, or (bb) T²¹³⁰→

C, under conditions of sufficient stringency to exclude hybridizations with (a) the sequence for a rat GlyT2 transporter or (b) the sequence for a mammalian GlyT1 transporter.

74. The nucleic acid sequence of claim 73, wherein the nucleic acid is a PCR primer and the stringent conditions are PCR conditions effective to amplify a human Glyt2 sequence but not to amplify (a) the sequence for a rat GlyT2 transporter or (b) the sequence for a mammalian GlyT1 transporter.

76. The antisense molecule of claim 73, wherein the contiguous stretch is included in the coding or non-coding strand of the nucleic acid sequence of SEQ ID NO:

121 or of a sequence that varies from the nucleic acid sequence of SEQ ID NO;

121 by having one or more of the following nucleotide substitutions;

(a) C⁷⁷→

T, (b) C²²⁰→

T, (c) C²²⁴→

T, (d) C²⁶⁶→

T, (e) A³⁰⁴→

G, (f) T⁵²¹→

A, (g) T⁵⁸³→

C, (h) A⁵⁹⁶→

G, (i) A⁶⁷⁸→

G, (j) T⁶⁸¹→

C, (k) G⁷⁴⁵→

T, (l) G⁷⁵⁰→

C, (m) C⁷⁶⁵→

T, (n) G⁷⁷⁷→

C, (o) A⁸⁶⁷→

G, (p) T⁹¹⁷→

C, (q) G¹²⁵⁶→

A, (r) T¹²⁹²→

C, (s) C¹³²⁵→

A, (t) C¹³⁶⁴→

A, (u) G¹³⁷⁴→

C, (v) C¹³⁹²→

A, (w) T¹⁴⁵⁴→

C, (x) A¹⁴⁷⁸→

G (y) T¹⁸⁵⁴→

C, (z) T¹⁹⁴⁹→

A, (aa) T¹⁹⁵⁹→

C, or (bb) T²¹³⁰→

C.

77. An expression vector comprising the nucleic acid of claim 73.

78. A method of reducing GlyT2 expression in a tissue or cell comprising applying to the tissue or cell (a) a nucleic acid of claim 73 in an amount effective to reduce GlyT2 expression or (b) an expression vector for expressing the nucleic acid in the tissue or cell in an amount effective to reduce GlyT2 expression.

79. A method of treating a nervous system disorder or condition comprising applying to a tissue or cell of a human patient a nervous system disorder- or condition-treating effective amount of a nucleic acid of claim 73 or a nervous system disorder- or condition-treating effective amount of an expression vector for expressing the nucleic acid in the tissue or cell.

75. A nucleic acid of at least about eighteen nucleotides in length comprising a contiguous sequence from the coding or non-coding strand of a human GlyT2 gene or cDNA, wherein the contiguous sequence has at least 1 nucleotide difference when compared with the rat GlyT2 gene sequence that aligns with the contiguous sequence.

80. A method for detecting whether an animal has autoimmune antibodies against a glycine transporter, the method comprising contacting an antibody preparation from the animal or a body fluid from the animal with a polypeptide antigen comprising a glycine transporter or derived from the glycine transporter.