Identification of transglutaminase substrates and uses therefor

Patent metadata

Field	Value
Publication number	US-11268120-B2
Application number	US-201715625784-A
Country	US
Kind code	B2
Filing date	Jun 16, 2017
Priority date	Dec 19, 2014
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

According to one aspect, the present disclosure provides a method of identifying a substrate of a transglutaminase using a peptide array comprising a plurality of peptides. The method includes the steps of contacting the peptides in the peptide array with the transglutaminase, allowing the transglutaminase to bind to the peptides, and identifying the substrate of the transglutaminase.

First claim

What is claimed is:

1. A method of cross-linking at least two compounds, the method comprising:
cross-linking a heterologous transglutaminase glutamine substrate peptide present in a first compound with a lysine substrate present in a second compound by contacting the heterologous transglutaminase glutamine substrate peptide and the lysine substrate with a microbial transglutaminase,
wherein:
the heterologous transglutaminase glutamine substrate peptide comprises the sequence motif [YF][VA]LQG and specifically binds the microbial transglutaminase; and
the microbial transglutaminase is a Streptomyces mobaraensis transglutaminase.

2. The method of claim 1 , wherein the heterologous transglutaminase glutamine substrate peptide comprises the sequence motif GDYALQGPG (SEQ ID NO: 79).

3. The method of claim 1 , wherein the lysine substrate is a heterologous transglutaminase lysine substrate peptide, the method further comprising the steps of incorporating the heterologous transglutaminase glutamine substrate peptide into the first compound and the heterologous transglutaminase lysine substrate peptide into the second compound.

4. The method of claim 3 , wherein the heterologous transglutaminase lysine substrate peptide comprises a sequence motif selected from SK[LS]K and [KR][ST]KL.

5. The method of claim 1 , wherein at least one of the first compound and the second compound is a Vitamin D binding protein.

6. The method of claim 3 , wherein the first compound is a Vitamin D binding protein, and wherein the heterologous transglutaminase glutamine substrate peptide comprises the sequence GGGGDYALQGGGG (SEQ ID NO: 85).

7. The method of claim 6 , wherein the second compound comprises a label, and wherein the heterologous transglutaminase lysine substrate peptide is incorporated into the label.

8. The method of claim 7 , wherein the label with the incorporated heterologous transglutaminase lysine substrate peptide is a compound with a formula selected from the group consisting of:

9. A method of cross-linking at least two compounds, the method comprising:
cross-linking a heterologous transglutaminase glutamine substrate peptide present in a first compound with a lysine substrate present in a second compound by contacting the heterologous transglutaminase glutamine substrate peptide and the lysine substrate with a microbial transglutaminase,
wherein:
the heterologous transglutaminase glutamine substrate peptide comprises the sequence motif [YF][VA]LQG and specifically binds the microbial transglutaminase; and
the microbial transglutaminase is a Streptoverticillium sp. transglutaminase.

10. The method of claim 1 , wherein the first compound is a protein comprising an N-terminus and a C-terminus and the heterologous transglutaminase glutamine substrate peptide is attached to the N-terminus or the C-terminus of the protein.

11. The method of claim 1 , wherein the glutamine substrate peptide comprises the sequence motif selected from the group consisting of GGGDYALQGGG (SEQ ID NO:26), CGGDYALQGPG (SEQ ID NO:27), WGGDYALQGPG (SEQ ID NO:28), YGGDYALQGPG (SEQ ID NO:29), DGGDYALQGPG (SEQ ID NO:30), GDGDYALQGPG (SEQ ID NO:31), NGGDYALQGPG (SEQ ID NO:32), GCGDYALQGPG (SEQ ID NO:33), EGGDYALQGPG (SEQ ID NO:34), PGGDYALQGPG (SEQ ID NO:35), TGGDYALQGPG (SEQ ID NO:36), QGGDYALQGPG (SEQ ID NO:37), IGGDYALQGPG (SEQ ID NO:38), FGGDYALQGPG (SEQ ID NO:39), HGGDYALQGPG (SEQ ID NO:40), LGGDYALQGPG (SEQ ID NO:41), VGGDYALQGPG (SEQ ID NO:42), RGGDYALQGPG (SEQ ID NO:43), GWGDYALQGPG (SEQ ID NO:44), MGGDYALQGPG (SEQ ID NO:45), SGGDYALQGPG (SEQ ID NO:46), AGGDYALQGPG (SEQ ID NO:47), GYGDYALQGPG (SEQ ID NO:48), GEGDYALQGPG (SEQ ID NO:49), GPGDYALQGPG (SEQ ID NO:50), GHGDYALQGPG (SEQ ID NO:51), WDGDYALQGGG (SEQ ID NO:52), GGGGDYALQGGGG (SEQ ID NO: 85), GGGDYALQGGGG (SEQ ID NO: 86), and GNGDYALQGPG (SEQ ID NO: 53), and a combination thereof.

12. The method of claim 11 , wherein the lysine substrate comprises a sequence motif selected from the group consisting of ARSKL (SEQ ID NO:54), KSKLA (SEQ ID NO:55), TKSKL (SEQ ID NO:56), KLSKL (SEQ ID NO:57), RSKLG (SEQ ID NO:58), RGSKL (SEQ ID NO:59), RGTKL (SEQ ID NO:60), FPKLK (SEQ ID NO:61), RSKSK (SEQ ID NO:62), SKSKL (SEQ ID NO:63), FTKSK (SEQ ID NO:64), KLKYK (SEQ ID NO:65), PKTKL (SEQ ID NO:66), RLKSK (SEQ ID NO:67), RSKLA (SEQ ID NO:68), GRSKL (SEQ ID NO:69), RAKYK (SEQ ID NO:70), SKLSK (SEQ ID NO:71), KLGAK (SEQ ID NO:72), QRSKL (SEQ ID NO:73), KTKYK (SEQ ID NO:74), LSKLK (SEQ ID NO:75), NRTKL (SEQ ID NO:76), QRTKL (SEQ ID NO:77), GGGRSKLAGGG (SEQ ID NO: 82), GGGARSKLGGGG (SEQ ID NO: 80), and GYKLK (SEQ ID NO:78), and a combination thereof.

13. The method of claim 1 , wherein the lysine substrate comprises a sequence motif selected from the group consisting of ARSKL (SEQ ID NO:54), KSKLA (SEQ ID NO:55), TKSKL (SEQ ID NO:56), KLSKL (SEQ ID NO:57), RSKLG (SEQ ID NO:58), RGSKL (SEQ ID NO:59), RGTKL (SEQ ID NO:60), FPKLK (SEQ ID NO:61), RSKSK (SEQ ID NO:62), SKSKL (SEQ ID NO:63), FTKSK (SEQ ID NO:64), KLKYK (SEQ ID NO:65), PKTKL (SEQ ID NO:66), RLKSK (SEQ ID NO:67), RSKLA (SEQ ID NO:68), GRSKL (SEQ ID NO:69), RAKYK (SEQ ID NO:70), SKLSK (SEQ ID NO:71), KLGAK (SEQ ID NO:72), QRSKL (SEQ ID NO:73), KTKYK (SEQ ID NO:74), LSKLK (SEQ ID NO:75), NRTKL (SEQ ID NO:76), QRTKL (SEQ ID NO:77), GGGRSKLAGGG (SEQ ID NO: 82), GGGARSKLGGGG (SEQ ID NO: 80), and GYKLK (SEQ ID NO:78), and a combination thereof.

14. A method of cross-linking at least two compounds, the method comprising the steps of:
cross-linking a heterologous transglutaminase glutamine substrate peptide present in a first compound with a lysine substrate present in a second compound,
wherein:
the cross-linking comprises allowing the heterologous transglutaminase glutamine substrate peptide and the lysine substrate to come in contact with the microbial transglutaminase whereby the microbial transglutaminase catalyzes the formation of an isopeptide bond between a γ-carboxamide group of a glutamine residue in the sequence motif [YF][VA]LQG and an ε-amino group of a lysine residue in the lysine substrate; and
the microbial transglutaminase is a Streptomyces mobaraensis transglutaminase.

15. The method of claim 14 , wherein the heterologous transglutaminase glutamine substrate peptide comprises the sequence motif selected from the group consisting of GGGDYALQGGG (SEQ ID NO:26), CGGDYALQGPG (SEQ ID NO:27), WGGDYALQGPG (SEQ ID NO:28), YGGDYALQGPG (SEQ ID NO:29), DGGDYALQGPG (SEQ ID NO:30), GDGDYALQGPG (SEQ ID NO:31), NGGDYALQGPG (SEQ ID NO:32), GCGDYALQGPG (SEQ ID NO:33), EGGDYALQGPG (SEQ ID NO:34), PGGDYALQGPG (SEQ ID NO:35), TGGDYALQGPG (SEQ ID NO:36), QGGDYALQGPG (SEQ ID NO:37), IGGDYALQGPG (SEQ ID NO:38), FGGDYALQGPG (SEQ ID NO:39), HGGDYALQGPG (SEQ ID NO:40), LGGDYALQGPG (SEQ ID NO:41), VGGDYALQGPG (SEQ ID NO:42), RGGDYALQGPG (SEQ ID NO:43), GWGDYALQGPG (SEQ ID NO:44), MGGDYALQGPG (SEQ ID NO:45), SGGDYALQGPG (SEQ ID NO:46), AGGDYALQGPG (SEQ ID NO:47), GYGDYALQGPG (SEQ ID NO:48), GEGDYALQGPG (SEQ ID NO:49), GPGDYALQGPG (SEQ ID NO:50), GHGDYALQGPG (SEQ ID NO:51), WDGDYALQGGG (SEQ ID NO:52), GGGGDYALQGGGG (SEQ ID NO: 85), GGGDYALQGGGG (SEQ ID NO: 86), and GNGDYALQGPG (SEQ ID NO: 53), and a combination thereof.

16. The method of claim 15 , wherein the lysine substrate comprises a sequence motif selected from the group consisting of ARSKL (SEQ ID NO:54), KSKLA (SEQ ID NO:55), TKSKL (SEQ ID NO:56), KLSKL (SEQ ID NO:57), RSKLG (SEQ ID NO:58), RGSKL (SEQ ID NO

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Albert Thomas
• Bergmann Frank
• Lyamichev Victor
• Patel Jigar
• Schraeml Michael
• Steffen Wojtek

Classifications

• C07K1/1075 Primary

  by covalent attachment of amino acids or peptide residues · CPC title

• C07K1/13

  Labelling of peptides · CPC title

• C07K14/4717

  Plasma globulins, lactoglobulin · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title