The lack of consensus for I-A(g7)-peptide binding motifs: is there a requirement for anchor amino acid side chains?

Abstract

We discuss here the problems in identifying sequence motifs of peptides that bind to I-A(g7), the class II histocompatibility molecule of NOD diabetic mice. We present studies that indicate a minor contribution of amino acid side chains for binding. A peptide from the Ealpha chain binds to I-A(g7) molecules and is recognized by CD4 T cells. By producing single-residue mutations we identified four residues that were considered to contact the T cell receptor. No residue was found to be essential for binding to I-A(g7): a peptide that contained the T cell contact residues, on a backbone of alanines, bound to I-A(g7) and stimulated the T cells. We conclude that peptides can bind to I-A(g7) without the requirement for residues with prominent side chains to anchor them.

Figure 1

Alanine scanning of 52–68 residues from Eα peptide. Different concentrations of alanine-substituted peptides were incubated in the presence of 5 × 10⁴ H12.25 T cell hybridoma and 5 × 10⁴ C3.G7 cells for 24 hr. Later, 100 μl of the supernatants was removed and assayed for IL-2 production by using 5 × 10³ CTLL-2 cells and incubated for 24 hr. Cells then were pulsed with 1 μCi/ml of [³H]thymidine for 16 hr. Two representative experiments are shown. (a) The T cell response of H12.25 hybridoma in the presence of the following alanine-substituted peptides: (●) wild-type 52–68, (○) Ala-53, (■) Ala-54, (□) Ala-55, (▴) Ala-57, and (▵) Ala-58. (b) The T cell response of H12.25 hybridoma in the presence of the following alanine-substituted peptides: (●) wild-type 52–68, (○) Ala-60, (■) Ala-62, (□) Ala-63, (▴) Ala-65, and (▵) Ala-66.

Figure 2

Alanine-substituted peptides: Ala-55, Ala-60, Ala-62, and Ala-63 can bind to I-A^g7 specifically. Different concentrations of alanine-substituted Eα 52–68 peptide (Ala-55, Ala-60, Ala-62, and Ala-63) or an unrelated nonbinding control peptide (HEL 48–61) were assayed for its ability to compete with OVA 323–339 peptide and block the T cell response of OVA-1 T cell clone. Competitor peptides plus 1 μM of 323–339 OVA peptide were incubated in the presence of 5 × 10³ OVA T cells and 5 × 10³ C3.G7 irradiated cells for 24 hr. Later, 25 μl of culture supernatant was removed and tested for IL-3 production by using FDCP3 cells as indicator cells.

Figure 3

A 15-mer polyalanine peptide is able to compete for binding to I-Ag7 with the 323–339 OVA peptide. (Left) A competition assay using different concentrations of a polyalanine peptide containing residues Glu-55, Leu-60, Asn-62, Ile-63 (●) or with a nonbinder peptide (○) 48–61 HEL. (Right) A competition assay using different concentrations of a 15-mer polyalanine peptide with an arginine at the carboxyl terminus to improve solubility (●). The results with the nonbinder peptide (○) 48–61 HEL from the left panel are included. Competitor peptides were tested as detailed in the legend of Fig. 2.

Figure 4

A polyalanine peptide containing T cell contact residues: Glu-55, Leu-60, Asn-62, and Ile-63 can stimulate H12.25 T cell hybridoma. Different concentrations of a polyalanine peptide containing residues Glu-55, Leu-60, Asn-62, and Ile-63 (○) or wild-type 52–68 Ea-peptide (●) were incubated in the presence of T cell hybridoma H12.25, and production of IL-2 was measured 24 hr later.