Application of an N-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine-Substituted Peptide as a Heterobifunctional Cross-Linking Agent in A Study Of Protein O-glycosylation in Yeast

Peter J. Crocker; David S. Watt; Richard R. Drake; Margaret Abramova; Crislyn D'Souza; Alan D. Elbein; James T. Slama; Katherine A. Wall

doi:10.1021/bc00013a011

Application of an N-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine-Substituted Peptide as a Heterobifunctional Cross-Linking Agent in A Study Of Protein O-glycosylation in Yeast

Peter J. Crocker, David S. Watt, Richard R. Drake, Margaret Abramova, Crislyn D'Souza, Alan D. Elbein, James T. Slama, Katherine A. Wall

Molecular and Cellular Biochemistry

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [¹²⁵I]-AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-Thr-Ser-Val ([¹²⁵I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with O-mannosyltransferase preparations, the hexapeptide was an acceptor of [¹⁴C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [¹²⁵I] azidoTyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the Ar-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.

Original language	English
Pages (from-to)	69-73
Number of pages	5
Journal	Bioconjugate Chemistry
Volume	3
Issue number	1
DOIs	https://doi.org/10.1021/bc00013a011
State	Published - Jan 1 1992

Bibliographical note

Funding Information:
We thank the National Science Foundation (CHE- 8607441 to D.S.W.), the National Institutes of Health (DK 21800toA.D.E.),andtheUniversityofKentuckyResearch Committee for the purchase of GC and HPLC instru- mentation (for D.S.W.).

Funding

Funders	Funder number
National Institute of Diabetes and Digestive and Kidney Diseases	R01DK021800

ASJC Scopus subject areas

Biotechnology
Bioengineering
Biomedical Engineering
Pharmacology
Pharmaceutical Science
Organic Chemistry

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10.1021/bc00013a011

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Crocker, P. J., Watt, D. S., Drake, R. R., Abramova, M., D'Souza, C., Elbein, A. D., Slama, J. T., & Wall, K. A. (1992). Application of an N-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine-Substituted Peptide as a Heterobifunctional Cross-Linking Agent in A Study Of Protein O-glycosylation in Yeast. Bioconjugate Chemistry, 3(1), 69-73. https://doi.org/10.1021/bc00013a011

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title = "Application of an N-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine-Substituted Peptide as a Heterobifunctional Cross-Linking Agent in A Study Of Protein O-glycosylation in Yeast",

abstract = "In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [125I]-AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-Thr-Ser-Val ([125I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with O-mannosyltransferase preparations, the hexapeptide was an acceptor of [14C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [125I] azidoTyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the Ar-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.",

author = "Crocker, {Peter J.} and Watt, {David S.} and Drake, {Richard R.} and Margaret Abramova and Crislyn D'Souza and Elbein, {Alan D.} and Slama, {James T.} and Wall, {Katherine A.}",

note = "Funding Information: We thank the National Science Foundation (CHE- 8607441 to D.S.W.), the National Institutes of Health (DK 21800toA.D.E.),andtheUniversityofKentuckyResearch Committee for the purchase of GC and HPLC instru- mentation (for D.S.W.).",

year = "1992",

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doi = "10.1021/bc00013a011",

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Application of an N-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine-Substituted Peptide as a Heterobifunctional Cross-Linking Agent in A Study Of Protein O-glycosylation in Yeast. / Crocker, Peter J.; Watt, David S.; Drake, Richard R. et al.
In: Bioconjugate Chemistry, Vol. 3, No. 1, 01.01.1992, p. 69-73.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Application of an N-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine-Substituted Peptide as a Heterobifunctional Cross-Linking Agent in A Study Of Protein O-glycosylation in Yeast

AU - Crocker, Peter J.

AU - Watt, David S.

AU - Drake, Richard R.

AU - Abramova, Margaret

AU - D'Souza, Crislyn

AU - Elbein, Alan D.

AU - Slama, James T.

AU - Wall, Katherine A.

N1 - Funding Information: We thank the National Science Foundation (CHE- 8607441 to D.S.W.), the National Institutes of Health (DK 21800toA.D.E.),andtheUniversityofKentuckyResearch Committee for the purchase of GC and HPLC instru- mentation (for D.S.W.).

PY - 1992/1/1

Y1 - 1992/1/1

N2 - In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [125I]-AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-Thr-Ser-Val ([125I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with O-mannosyltransferase preparations, the hexapeptide was an acceptor of [14C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [125I] azidoTyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the Ar-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.

AB - In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [125I]-AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-Thr-Ser-Val ([125I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with O-mannosyltransferase preparations, the hexapeptide was an acceptor of [14C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [125I] azidoTyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the Ar-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.

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Application of an N-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)tyrosine-Substituted Peptide as a Heterobifunctional Cross-Linking Agent in A Study Of Protein O-glycosylation in Yeast

Abstract

Bibliographical note

Funding

ASJC Scopus subject areas

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