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.
In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [125I]-N-(4-azido-2,3,5,6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-T hr-Ser-Val ([125I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, N-(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 N-(4-azido-2,3,5,6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.
Drake
R R
RR
Department of Chemistry, University of Kentucky, Lexington 40506.
Slama
J T
JT
Wall
K A
KA
Abramova
M
M
D'Souza
C
C
Elbein
A D
AD
Crocker
P J
PJ
Watt
D S
DS
D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents; D005656:Fungal Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D008364:Mannosyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D006023:Glycoproteins; C075480:N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine; D014443:Tyrosine; D008364:Mannosyltransferases