TFF2 Human, His

What is TFF2 and what distinguishes it structurally?

TFF2 (also known as Spasmolytic polypeptide, Spasmolysin, or SML1) belongs to the trefoil factor family of proteins characterized by the trefoil motif, a 40-amino acid domain containing three conserved disulfide bonds. Human TFF2 is a secretory protein expressed predominantly in gastrointestinal mucosa. The recombinant human TFF2 with His tag is a single, non-glycosylated polypeptide chain containing 116 amino acids (positions 24-129) with a 10-amino acid His tag fused at the N-terminus, resulting in a total molecular mass of 13.2 kDa . The non-tagged version contains 106 amino acids (positions 24-129) with a molecular mass of approximately 12 kDa .

What are the primary biological functions of TFF2?

TFF2 serves several critical functions in the gastrointestinal tract. It protects the mucosa from various insults, stabilizes the mucus layer through interactions with carbohydrate side chains of glycoproteins, and promotes healing of the epithelium. Additionally, TFF2 inhibits gastric acid motility and secretion . Recent research has uncovered previously unsuspected roles for TFF2 in energy balance regulation, suggesting it may be a central regulator in metabolic processes and a potential therapeutic target for obesity . TFF2 also demonstrates immunomodulatory properties, with suppressive effects on proinflammatory cytokine production in certain contexts .

Where is TFF2 primarily expressed in humans?

In normal human physiology, TFF2 is predominantly expressed in the mucous neck cells of the gastric fundus and in glands at the base of the gastric antrum. It is secreted into gastric juice in significant quantities . TFF2 can also be detected at elevated concentrations around sites of ulceration, consistent with its tissue-protective and repair functions . While normally expressed at low levels in other gastrointestinal tissues, TFF2 expression is upregulated in certain cancer tissues, including those of the pancreas, colon, and bile ducts .

How should recombinant TFF2 be reconstituted and stored for optimal stability?

For recombinant His-tagged TFF2:

• Reconstitute the lyophilized protein with deionized water to approximately 0.5 mg/ml

• Allow complete dissolution of the lyophilized pellet

• Filter through an appropriate sterile filter before cell culture applications

• For short-term storage (2-7 days), store at 4°C

• For long-term storage, keep desiccated below -18°C

• Add carrier protein (0.1% HSA or BSA) for extended storage stability

• Avoid freeze-thaw cycles

For non-tagged recombinant TFF2:

• Reconstitute in sterile 18MΩ-cm H₂O at a concentration not less than 100 μg/ml

• This can be further diluted to other aqueous solutions as needed

• Follow the same storage recommendations as for His-tagged TFF2

What experimental conditions affect TFF2 expression in cell culture systems?

Research has shown that microenvironmental conditions significantly influence TFF2 expression. pH appears to be a particularly critical factor. Experimental data demonstrates that acidic conditions dramatically upregulate TFF2 expression:

• At pH 6.5: 42.8-fold increase in expression compared to pH 7.4

• At pH 6.8: 5.8-fold increase in expression compared to pH 7.4

Temperature modulation (increasing from 37°C to 40°C) did not induce TFF2 expression in experimental settings. This pH-dependent expression pattern may reflect TFF2's role in protecting cells against acidic environments, both in normal physiological contexts and in tumor microenvironments, which typically exhibit lower pH (6.2-6.9) compared to normal tissues (pH 7.2-7.4) .

How does glycosylation affect human TFF2 and what are the implications for experimental design?

Human TFF2 undergoes N-linked glycosylation in vivo, presumably at Asn15, which forms part of the single consensus site for N-glycosylation in the protein. This post-translational modification produces a higher molecular weight form that can be detected alongside the 12 kDa non-glycosylated form in gastric juice and mucosal tissues. The majority of TFF2 in normal gastric mucosa appears to be glycosylated .

For researchers using recombinant TFF2, it's important to note that E. coli-produced proteins lack this glycosylation. When comparing experimental results with physiological functions, this difference should be considered, as glycosylation may have functional significance. Experimentally, the glycosylation status can be confirmed using endoglycosidase treatment (peptide-N-glycosidase F) .

What is the role of TFF2 in metabolic regulation and energy homeostasis?

Studies using TFF2 knockout (KO) mice have revealed surprising roles in energy balance regulation. When challenged with a high-fat diet, TFF2 KO mice demonstrated:

• Greater appetite and higher energy intake compared to wild-type mice

• Lower serum leptin levels

• Increased transcription of agouti-related protein (Agrp) in the hypothalamus

• Enhanced energy and triglyceride fecal excretion

• Protection against high-fat diet-induced obesity

• Reduced weight gain and fat depot accumulation while maintaining normal lean mass

• Lower energy efficiency

• Increased energy expenditure and locomotor activity

These findings suggest TFF2 functions as a significant regulator of energy balance, potentially through modulation of appetite-regulating hormones and central nervous system pathways controlling food intake.

How does TFF2 interact with immune responses in parasitic infections?

Research examining helminth infections in human populations has provided insights into TFF2's immunomodulatory roles:

• In a Brazilian cohort, hookworm infection preferentially elevated TFF2 levels, with a stronger positive correlation with age rather than parasite burden

• In Nigerian children with Schistosoma haematobium infection, serum TFF2 and TFF3 levels were decreased, corresponding with higher levels of cytokines in urine

• These cytokines included the type 2 cytokine IL-13, proinflammatory cytokines TNFα and IL-1β, and the regulatory cytokine IL-10

• Experimental exposure of human PBMCs to TFF2 (but not TFF3) suppressed PHA-induced proinflammatory cytokine production (TNFα and IFN-γ)

These observations suggest that TFF2 may contribute to immunoregulation during parasitic infections, possibly promoting an environment that balances protective immunity with tissue protection.

What discrepancies exist between human and mouse TFF2 biological functions?

Important differences have been observed between mouse and human TFF2 biology that researchers should consider:

Understanding these discrepancies is crucial for translating findings from mouse models to human applications and may identify targets for improving anti-helminth immunity.

What are optimal protocols for measuring TFF2 levels in biological samples?

Based on published methodologies, researchers have successfully quantified TFF2 using:

• Quantitative western transfer analysis for gastric juice and mucosal cytosol samples

• ELISA assays for detecting TFF2 in serum and other body fluids

• RT-PCR followed by relative quantification for measuring TFF2 gene expression in cells

When performing these analyses, researchers should be aware that:

• Both glycosylated and non-glycosylated forms of TFF2 may be present

• Antibodies should be raised against the correct amino acid sequence

• Glycosylation status can be determined through endoglycosidase treatment (PNGase F)

How can TFF2's effects on immune cell function be experimentally assessed?

Experimental approaches to study TFF2's immunomodulatory properties include:

• PBMC isolation and culture:

  • Plate 2.5 × 10⁵ cells per well in RPMI buffer containing 5-10% human non-autologous plasma

  • Maintain in 37°C incubation with 5% CO₂

• Treatment protocol:

  • Pre-treat cells with human recombinant TFF2 overnight (25 ng/μL)

  • Stimulate with phytohemagglutinin (PHA, 50μg) for 24 hours

  • Include unstimulated controls both with and without exogenous TFF2

• Analysis:

  • Collect supernatants

  • Measure cytokines (IFNγ, TNFα, etc.) by ELISA

  • Run each treatment point in duplicate

This methodology has successfully demonstrated TFF2's suppressive effects on proinflammatory cytokine production by human immune cells.

How might TFF2's pH-dependent expression be relevant for cancer research?

The finding that TFF2 expression is dramatically upregulated under acidic conditions may have significant implications for cancer research. Tumor microenvironments typically exhibit lower pH (6.2-6.9) compared to normal tissues (pH 7.2-7.4), and TFF2 is highly expressed in various cancer tissues including colon, pancreatic, and bile duct cancers .

This suggests TFF2 may serve a protective function for cancer cells in acidic environments. Further research directions could include:

• Investigating whether TFF2 knockdown sensitizes cancer cells to acidic pH

• Exploring TFF2 as a biomarker for tumors with particularly acidic microenvironments

• Developing therapeutic approaches targeting TFF2 in cancer contexts

What are the most promising therapeutic applications for TFF2 research?

Based on current research, several therapeutic directions appear promising:

• Obesity management: Given TFF2's role in energy balance regulation, it represents a potential therapeutic target for obesity. The protective effect of TFF2 KO against high-fat diet-induced obesity suggests that TFF2 inhibition might have therapeutic potential .

• Mucosal protection: TFF2's natural role in mucosal protection and healing suggests potential applications in treating inflammatory conditions of the gastrointestinal tract.

• Immunomodulation: TFF2's ability to suppress proinflammatory cytokine production suggests potential applications in inflammatory disorders. Further research is needed to develop appropriate delivery systems and understand long-term effects .