The I-Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder.

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes.

Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position beta57. I-Ag7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-Ag7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-Ag7 has a substantially wider peptide-binding groove around beta57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp(beta57) leads to an oxyanion hole in I-Ag7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.

Bacterial expression and purification of recombinant Plasmodium yoelii circumsporozoite protein.

We report the expression and purification of recombinant rodent malarial Plasmodium yoelii circumsporozoite surface protein (PyCSP) in Escherichia coli. To facilitate purification of the recombinant protein, the PyCSP was expressed as an amino-terminal fusion protein to glutathione S-transferase and as a carboxy-terminal fusion protein to a hexahistidyl tag. The expression of the fusion protein was controlled by the inducible tac promoter. Under optimal conditions the immunoreactive PyCSP represented approximately 0.04% of the total cell lysate. Western blot analysis probing with an anti-PyCSP antibody revealed a wide array of immunoreactive bands. Material isolated by affinity purification on glutathione-Sepharose 4B resin also contained multiple bands indicative of premature termination or carboxyl-terminal degradation. Analysis of protein retained on a nickel nitrilotriacetic acid resin revealed evidence of amino-terminal deleted material. Combining the two mild affinity purifications resulted in isolation of a single immunoreactive protein of approximate molecular weight of 96 kDa. We anticipate that the approach described in this study will facilitate the production of highly purified recombinant proteins.

Elemental and trace element distribution in medical samples: analysis by proton-induced X-ray emission.

The analysis of trace elements is performed by proton-induced X-ray emission. The process is most effective if the velocity of the exciting particles--protons--is similar to the velocity of the electron on its orbit in the simple atomic model of Bohr. For K-shell electrons of the elements with 15 less than or equal to z less than or equal to 40 this requires proton energies of a few MeV, available from electrostatic van de Graaf accelerator machines. After knocking out the K-shell electron, the empty place is filled up by electrons jumping from higher orbits with simultaneous emission of characteristic X-rays, which are registered with a cooled Si (Li) detector. By a set of electrodes the beam can be swept across the specimen surface. Therefore this method yields an excellent correlation of trace element distribution within the morphological structure of organic tissue. In the present study the sweep went along a line perpendicular to the arterial wall layers (aortic, renal artery and heart muscle) of normotensive and spontaneously hypertensive rats. Along this line all elements and trace elements are recorded simultaneously. These are P, S, Cl, K, Ca, Fe, Cu, Zn, Br and Sr. The trace element content of the aortic wall and the renal artery, of 22 spontaneously hypertensive and 11 normotensive rats and of human heart muscle was investigated. The results demonstrate that Zn was only detected in the muscle-containing layers of the arteries. There was no different distribution between hypertensive and normotensive rats. However, Ca2+ was mainly detected in the smooth muscle-containing tunica media of hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)