The human two domain trefoil protein, TFF2, is glycosylated in vivo in the stomach.
BACKGROUND
TFF2, a member of the trefoil factor family (TFF) of peptides, is a secreted protein of 106 amino acids that is expressed in mucous neck cells of the fundus and glands at the base of the antrum in normal human stomach. TFF2 is also detected at high concentrations around sites of ulceration. It is protective against mucosal damaging agents and stimulates cell motility.


OBJECTIVE
To measure the expression of TFF2 in normal human stomach and its secretion into gastric juice.


METHODS
TFF2 cDNA was amplified by reverse transcription polymerase chain reaction from gastric mucosa and sequenced. Gastric juice or cytosol, prepared from gastric mucosa, was obtained from individuals with macroscopically normal stomachs. TFF2 concentrations were measured by quantitative western transfer analysis.


RESULTS
Sequencing of TFF2 cDNA revealed a single amino acid change from the published sequence. Significant amounts of 12 kDa TFF2 were detected in human gastric juice. Larger quantities of a protein of higher apparent molecular mass were also detected. This was shown to be N-glycosylated TFF2 using the endoglycosidase, peptide-N-Gycosidase F. The majority of TFF2 in normal gastric mucosa was also glycosylated.


CONCLUSIONS
Human TFF2 is glycosylated via an N-linkage, presumably on Asn(15) which forms part of the single consensus site for N-glycosylation in human TFF2. The glycosylation may be of functional significance. Future studies of human TFF2 should use antibodies raised against the correct amino acid sequence. Biological studies should be performed with recombinant protein of the correct sequence, and the biological consequences of glycosylation investigated.
May
F E
FE
Department of Pathology, School of Clinical and Laboratory Sciences, University of Newcastle-upon-Tyne, Royal Victoria Infirmary, Newcastle-upon-Tyne NE1 4LP, UK.
Semple
J I
JI
Newton
J L
JL
Westley
B R
BR
D018076:DNA, Complementary / Q000032:analysis; D005750:Gastric Juice / Q000737:chemistry*; D005753:Gastric Mucosa / Q000737:chemistry*; D006031:Glycosylation; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3
D018076:DNA, Complementary; D006133:Growth Substances; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides; C000604007:TFF2 protein, human; C099037:TFF3 protein, rat; C000604008:Tff2 protein, rat; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3