NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water

Iñigo Alaña,Jeremy C Parker,Victor A Gault,Peter R Flatt,Finbarr P.M O'Harte,J Paul G Malthouse,Chandralal M Hewage

doi:10.1074/jbc.m510414200

Iñigo Alaña, Jeremy C Parker + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m510414200

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Abstract

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases. In this study, we present the solution structure of GIP in water determined by NMR spectroscopy. The calculated structure is characterized by the presence of an alpha-helical motif between residues Ser(11) and Gln(29). The helical conformation of GIP is further supported by CD spectroscopic studies. Six GIP-(1-42)Ala(1-7) analogues were synthesized by replacing individual N-terminal residues with alanine. Alanine scan studies of these N-terminal residues showed that the GIP-(1-42)Ala(6) was the only analogue to show insulin-secreting activity similar to that of the native GIP. However, when compared with glucose, its insulinotropic ability was reduced. For the first time, these NMR and modeling results contribute to the understanding of the structural requirements for the biological activity of GIP.

Highlights

It is clear that native Glucose-dependent insulinotropic polypeptide (GIP) dose-dependently (10Ϫ12-10Ϫ6 M) stimulated insulin secretion when compared with control incubations (5.6 mM glucose alone) (p Ͻ 0.001)
GIP-(1– 42)Ala6 stimulated insulin secretion when compared with control (p Ͻ 0.01 to p Ͻ 0.001), not to the same extent as native GIP (p Ͻ 0.05 to p Ͻ 0.01)
Seven synthetic peptides with a singleresidue replacement with an alanine in each of the first seven positions were synthesized to evaluate specific residues that play a role in insulin secretion

Summary

EXPERIMENTAL PROCEDURES

Synthesis and Characterization of GIP and Its Analogues—Human GIP-(1– 42) and GIP-(1– 42)Ala, GIP-(1– 42)Ala, GIP-(1– 42)Ala, GIP-(1– 42)Ala, GIP-(1– 42)Ala, and GIP-(1– 42)Ala were sequentially synthesized on an Applied Biosystems 432A automated peptide synthesizer using standard solid-phase Fmoc procedure, starting from an Fmoc-Gln-Wang resin as described previously [17]. For studies in the far-UV, GIP samples were dissolved in acetate buffer (20 mM, pH 4.0, 5.0), phosphate buffer (20 mM, pH 6.0, 7.0, 8.0), or in water (unbuffered, pH 6.8, uncorrected) for a final peptide concentration of 30 ␮M. For the near-UV, GIP samples were dissolved in phosphate buffer (20 mM, pH 7.0) or in water (unbuffered, pH 3.0, uncorrected) for a final peptide concentration of 0.95 mM. The peptide concentration was determined using the UV absorbance at 280 nm and an extinction coefficient of 13,980 MϪ1 cmϪ1, as calculated with ProtParam [21]. Experiments in the far-UV were performed using a response of 2 s and a scanning speed of 50 nm/min, by accumulation of 15 scans.

NMR and Alanine Scan Studies of GIP

RESULTS

DISCUSSION

Structural statistics of GIP in water