PMID- 11258925
OWN - NLM
STAT- MEDLINE
DCOM- 20010503
LR - 20071114
IS - 0006-2960 (Print)
IS - 0006-2960 (Linking)
VI - 40
IP - 10
DP - 2001 Mar 13
TI - Characterization of the glycosylation sites in cyclooxygenase-2 using mass
spectrometry.
PG - 3109-16
AB - Cyclooxygenase is involved in the biosynthesis and function of prostaglandins. It
is a glycoprotein located in the endoplasmic reticulum and in the nuclear
envelope, and it has been found to have two isoforms termed COX-1 and COX-2. This
paper reports on the glycosylation site analysis of recombinant COX-2 using
matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass
spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The
nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average
molecular masses of 71.4, 72.7, and 73.9 kDa. Each glycoform contained a number
of peaks differing by 162 Da indicating heterogeneity and suggesting the presence
of high-mannose sugars. The masses of the glycoforms indicate that
oligosaccharides occupy two to four sites and a single N-acetylglucosamine
(GlcNAc) residue occupied up to two sites. The MALDI MS analysis of a tryptic
digest of the protein showed a number of potential glycopeptides. The peptides
differed by 162 Da which further suggested high-mannose sugars. Nanoelectrospray
MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and
confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg
587 + GlcNAc containing Asn 580. It was not possible to conclusively determine
whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic
digest was deglycosylated to confirm the presence of the glycopeptides. Finally,
a non-glycosylated tryptic peptide was observed containing the Asn 592.
FAU - Nemeth, J F
AU - Nemeth JF
AD - Mass Spectrometry Research Center, and Department of Biochemistry, Vanderbilt
University Medical Center, Nashville, Tennesee 37232-6400, USA.
FAU - Hochgesang, G P Jr
AU - Hochgesang GP Jr
FAU - Marnett, L J
AU - Marnett LJ
FAU - Caprioli, R M
AU - Caprioli RM
LA - eng
GR - 5R35 CA47479/CA/NCI NIH HHS/United States
GR - GM 58008-02/GM/NIGMS NIH HHS/United States
GR - T32 ES07028/ES/NIEHS NIH HHS/United States
PT - Journal Article
PT - Research Support, U.S. Gov't, P.H.S.
PL - United States
TA - Biochemistry
JT - Biochemistry
JID - 0370623
RN - 0 (Glycopeptides)
RN - 0 (Isoenzymes)
RN - 0 (Peptide Fragments)
RN - 7006-34-0 (Asparagine)
RN - EC 1.14.99.1 (Cyclooxygenase 2)
RN - EC 1.14.99.1 (Prostaglandin-Endoperoxide Synthases)
RN - EC 3.4.21.4 (Trypsin)
SB - IM
EIN - Biochemistry 2001 Jul 24;40(29):8606. Hochensang GP Jr [corrected to Hochgesang
GP Jr]
MH - Amino Acid Sequence
MH - Animals
MH - Asparagine/metabolism
MH - Cyclooxygenase 2
MH - Genetic Vectors/biosynthesis/chemical synthesis
MH - Glycopeptides/analysis/metabolism
MH - Glycosylation
MH - Isoenzymes/biosynthesis/*chemistry/genetics/*metabolism
MH - Mice
MH - Molecular Sequence Data
MH - Peptide Fragments/analysis/metabolism
MH - Prostaglandin-Endoperoxide Synthases/biosynthesis/*chemistry/genetics/*metabolism
MH - Spectrometry, Mass, Electrospray Ionization
MH - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
MH - Trypsin/metabolism
EDAT- 2001/03/22 10:00
MHDA- 2001/05/05 10:01
CRDT- 2001/03/22 10:00
PHST- 2001/03/22 10:00 [pubmed]
PHST- 2001/05/05 10:01 [medline]
PHST- 2001/03/22 10:00 [entrez]
AID - bi002313c [pii]
PST - ppublish
SO - Biochemistry. 2001 Mar 13;40(10):3109-16.
PMID- 9118998
OWN - NLM
STAT- MEDLINE
DCOM- 19970422
LR - 20161017
IS - 0014-2956 (Print)
IS - 0014-2956 (Linking)
VI - 244
IP - 2
DP - 1997 Mar 1
TI - Glycosylation analysis and protein structure determination of murine fetal
antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk),
preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs.
PG - 334-42
AB - By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus
musculus amniotic fluid was shown to be the circulating protein of the delta-like
protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1
(Pref-1) gene products. The protein contains 36 cysteine residues arranged in six
epidermal-growth-factor-like domains. The purification of several C-terminal
peptides of varying lengths showed mFA1 to be C-terminal heterogeneous. O-linked
glycosylations of the NeuNAc alpha2-3Gal beta1-3(NeuNAc alpha2-6)GalNAc type were
present on all C-terminal peptides at residues Thr235, Thr244 and Thr248,
although glycosylation on Thr244 was only partial. Three N-linked glycosylations
were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and
Asn151) were in the unusual Asn-Xaa-Cys motif. Fucosylated biantennary
complex-type and small amounts (less than 5%) of triantennary complex-type
structures were identified on the glycosylated asparagine residues using
sequential exoglycosidase and endoglycosidase digestions combined with
matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The
presence of O-linked monosaccharides (glucose attached to Ser71, Ser193 and
fucose at Thr201) was tentatively ascertained by combining Edman degradation and
MALDI-MS. The results presented shows mFA1 to be the circulating heterogeneous
cleavage products of the membrane-bound protein encoded by the murine cDNAs dlk,
pref-1 and SCP-1.
FAU - Krogh, T N
AU - Krogh TN
AD - Department of Molecular Biology, Odense University, Denmark.
FAU - Bachmann, E
AU - Bachmann E
FAU - Teisner, B
AU - Teisner B
FAU - Skjodt, K
AU - Skjodt K
FAU - Hojrup, P
AU - Hojrup P
LA - eng
PT - Journal Article
PT - Research Support, Non-U.S. Gov't
PL - England
TA - Eur J Biochem
JT - European journal of biochemistry
JID - 0107600
RN - 0 (Chemokine CXCL12)
RN - 0 (Chemokines)
RN - 0 (Chemokines, CXC)
RN - 0 (Cxcl12 protein, mouse)
RN - 0 (DNA, Complementary)
RN - 0 (Dlk1 protein, mouse)
RN - 0 (Glycoproteins)
RN - 0 (Intercellular Signaling Peptides and Proteins)
RN - 0 (Intracellular Signaling Peptides and Proteins)
RN - 0 (Membrane Proteins)
RN - 0 (Neoplasm Proteins)
RN - 0 (Peptide Fragments)
RN - 0 (Repressor Proteins)
RN - 0 (delta protein)
SB - IM
MH - Amino Acid Sequence
MH - Animals
MH - Carbohydrate Sequence
MH - Chemokine CXCL12
MH - Chemokines/*genetics
MH - *Chemokines, CXC
MH - DNA, Complementary/genetics
MH - Female
MH - Glycoproteins/blood/*chemistry/*genetics
MH - Glycosylation
MH - Intercellular Signaling Peptides and Proteins
MH - Intracellular Signaling Peptides and Proteins
MH - Membrane Proteins/*genetics
MH - Mice
MH - Molecular Sequence Data
MH - Molecular Structure
MH - Neoplasm Proteins/*genetics
MH - Peptide Fragments/chemistry/genetics/isolation & purification
MH - Pregnancy
MH - Repressor Proteins/*genetics
MH - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
EDAT- 1997/03/01 00:00
MHDA- 2000/03/25 09:00
CRDT- 1997/03/01 00:00
PHST- 1997/03/01 00:00 [pubmed]
PHST- 2000/03/25 09:00 [medline]
PHST- 1997/03/01 00:00 [entrez]
PST - ppublish
SO - Eur J Biochem. 1997 Mar 1;244(2):334-42.
PMID- 2108149
OWN - NLM
STAT- MEDLINE
DCOM- 19900503
LR - 20131121
IS - 0021-9258 (Print)
IS - 0021-9258 (Linking)
VI - 265
IP - 10
DP - 1990 Apr 5
TI - The polypeptide of immunoglobulin G influences its galactosylation in vivo.
PG - 5833-9
AB - To examine the nature of the factors influencing the galactosylation pattern of
the heavy chain of murine immunoglobulin G (IgG), cell fusion was performed
between a myeloma (P3x63Ag8) and a hybridoma (Sp2HL/Bu) cell line which secrete
different IgGs possessing structurally distinct CH2-linked oligosaccharide
moieties. The glycosylation patterns of the IgGs of the parental and fused cells
were studied. Pronase digestion of the purified heavy chains and subsequent end
labeling with fluorescein isothiocyanate produced fluoresceinated glycopeptides
which were detected and purified by polyacrylamide gel electrophoresis.
Structural information was obtained by enzymatic digestion, lectin affinity
chromatography, and methylation analysis. IgGs from both parental lines possessed
oligosaccharide units displaying microheterogeneity based upon a common
symmetrical biantennary structure terminating in beta-GlcNAc. The structures of
both IgGs, however, differed in the pattern of the mono- and digalactosylated
components. Clones, selected following the fusion of the parental cells, were
expanded; and the individual IgGs were purified. All clones produced homodimeric
IgG1 and IgG2b as well as heterodimeric IgG possessing both the gamma 1 and gamma
2b heavy chains. Analysis of the carbohydrate moieties of the gamma 1 chain from
the homodimeric and heterodimeric IgGs and of the gamma 2b chain from the
heterodimeric molecule demonstrates that the polypeptide structure of the heavy
chain influences the terminal galactosylation of the glycan unit at the conserved
site of glycosylation of IgGs.
FAU - Lee, S O
AU - Lee SO
AD - Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway,
New Jersey 08855-1059.
FAU - Connolly, J M
AU - Connolly JM
FAU - Ramirez-Soto, D
AU - Ramirez-Soto D
FAU - Poretz, R D
AU - Poretz RD
LA - eng
GR - AI 06471/AI/NIAID NIH HHS/United States
PT - Journal Article
PT - Research Support, Non-U.S. Gov't
PT - Research Support, U.S. Gov't, P.H.S.
PL - United States
TA - J Biol Chem
JT - The Journal of biological chemistry
JID - 2985121R
RN - 0 (Glycopeptides)
RN - 0 (Immunoglobulin G)
RN - 0 (Immunoglobulin Heavy Chains)
RN - 0 (Immunoglobulin gamma-Chains)
RN - 0 (Macromolecular Substances)
RN - EC 3.2.1.- (Glycoside Hydrolases)
RN - EC 3.2.1.52 (Acetylglucosaminidase)
RN - EC 3.4.24.- (Pronase)
RN - X2RN3Q8DNE (Galactose)
SB - IM
MH - Acetylglucosaminidase/metabolism
MH - Animals
MH - Carbohydrate Sequence
MH - Cloning, Molecular
MH - Galactose/*metabolism
MH - Glycopeptides/isolation & purification/metabolism
MH - Glycoside Hydrolases/metabolism
MH - Glycosylation
MH - Hybridomas/immunology
MH - Immunoglobulin G/*metabolism
MH - Immunoglobulin Heavy Chains/*metabolism
MH - Immunoglobulin gamma-Chains/analysis/metabolism
MH - Macromolecular Substances
MH - Methylation
MH - Mice
MH - Molecular Sequence Data
MH - Pronase/metabolism
MH - Tumor Cells, Cultured
EDAT- 1990/04/05 00:00
MHDA- 1990/04/05 00:01
CRDT- 1990/04/05 00:00
PHST- 1990/04/05 00:00 [pubmed]
PHST- 1990/04/05 00:01 [medline]
PHST- 1990/04/05 00:00 [entrez]
PST - ppublish
SO - J Biol Chem. 1990 Apr 5;265(10):5833-9.