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. 2000 Apr;46(4):454–459. doi: 10.1136/gut.46.4.454

The human two domain trefoil protein, TFF2, is glycosylated in vivo in the stomach

F May 1, J Semple 1, J Newton 1, B Westley 1
PMCID: PMC1727891  PMID: 10716671

Abstract

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.
AIMS—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 Asn15 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.


Keywords: TFF peptide; stomach; glycosylation; TFF2; restitution

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Figure 1  .

Figure 1  

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Sequence of TFF 2 protein. (A) Sequence of the mature TFF2 protein derived from TFF2 mRNA from human gastric mucosa of three individuals and the TFF2 gene. The positions of the two trefoil domains are indicated as are the amino acids included in each of the three loops formed during folding of the trefoil domains. The single N-glycosylation recognition sequence (NRT) which is a potential glycosylation site located in loop 1 of the first trefoil domain is boxed. The lysine residue (K) which replaces the asparagine residue found in the previously published sequence1 at position 99 is also boxed. The 16 amino acid peptide synthesised for use as an immunogen is overlined. Spaces have been inserted in the sequence after residues 6, 49, and 98 to indicate the limits of the trefoil domains. (B) The amino acid sequences of the first loop in domain I of the four known mammalian TFF2 proteins: human TFF21 (present study); porcine TFF225; murine TFF226; and rat TFF2.27 (C) Comparison of the 16 amino acid residues at the carboxy termini of the four known mammalian TFF2 protein sequences proteins: human TFF2 (present study); porcine TFF225; murine TFF226; and rat TFF2.27

Figure 2  .

Figure 2  

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Detection of TFF2 in human gastric juice. Aliquots (10 µl) of gastric juice were subjected to electrophoresis on a denaturing polyacrylamide gel, transferred to a PVDF membrane, and reacted with TFF2 antisera. Known amounts of recombinant TFF2 were also analysed. The positions of the molecular mass markers (MW) are shown on the left and of TFF2 on the right hand side.

Figure 3  .

Figure 3  

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Human TFF2 is glycosylated via an N-linkage. Aliquots of gastric juice were denatured and incubated under denaturing conditions in the absence or presence of peptide-N-Gycosidase F (PNGase F), and in the absence or presence of pepstatin. TFF2 was detected by western transfer analysis. The position of TFF2 is shown on the right hand side of the gel and of the molecular weight markers on the left hand side.

Figure 4  .

Figure 4  

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Detection of TFF2 in cytosol prepared from human gastric mucosa by western transfer analysis. Cytosol was prepared from small biopsy specimens of normal human gastric mucosa (samples 1-5), or mucosa from gastrectomy specimens (samples A-C). The positions of the molecular mass markers (MW) are shown on the left and of TFF2 on the right hand side.

Selected References

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