The newly identified TIM family of proteins is associated with regulation of T helper type 1 (T(H)1) and T(H)2 immune responses. TIM-1 is genetically linked to asthma and is a receptor for hepatitis A virus, but the endogenous ligand of TIM-1 is not known. Here we show that TIM-4, which is expressed by antigen-presenting cells, is the ligand for TIM-1. In vivo administration of either soluble TIM-1-immunoglobulin (TIM-1-Ig) fusion protein or TIM-4-Ig fusion protein resulted in hyperproliferation of T cells, and TIM-4-Ig costimulated T cell proliferation mediated by CD3 and CD28 in vitro. These data suggest that the TIM-1-TIM-4 interaction is involved in regulating T cell proliferation.

Urotensin I, purified from extracts of the urophysis of a teleost fish (Catostomus commersoni), exhibits potent hypotensive activity (mammals and birds) and corticotropin-releasing activity (both fish and mammals). The primary structure of this 41-residue peptide was determined to be H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Asn-Met-Ile-Glu- Met-Ala-Arg-Ile-Glu-Asn-Glu-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu -Val-NH2. Extraction with 0.1N HCl at 100 degrees C cleaves the amino-terminal tripeptide, yeilding a fully active analog, urotensin I(4-41). The amino acid sequence was confirmed by measuring the biological activity of synthetic urotensin I(4-41). Urotensin I exhibits a striking sequence homology with ovine corticotropin-releasing factor and with frog sauvagine. These three peptides exhibit similar activities in biological test systems.

The complete amino acid sequence was determined for bovine ubiquitin, and adenylate cyclase stimulating polypeptide, which is probably represented universally in living cells. Ubiquitin has a molecular weight of 8451 and consists of a single polypeptide chain containing 74 amino acid residues. It contains four arginine residues but no cysteine or trytophan residues. The first 61 amino acid residues were obtained by automated Edman degradations. Tryptic digestion of maleated ubiquitin yielded four peptide fragments that were resolved by molecular sieve chromatography and coded in order of decreasing chain length (MT-1, MT-2, MT-3, and MT-4). The automated sequenator determinations on native ubiquintin provided overlapping sequence data for three of these fragments that gave an order of MT-1, MT-3, and then MT-2; Peptide MT-4, a dipeptide, was therefore assigned to the C terminus, and the placement of peptide MT-2 was corroborated by analysis of data from carboxypeptidase digestions of maleated ubiquitin. Peptide MT-2 was domaleated and sequenced by manual Edman degradations through a single lysine residue. It was cleaved at this residue with trypsin, and the two resultant peptides were separated by ion-exchange chromatography. Manual sequencing of the C-terminal demaleated tryptic peptide of MT-2 completed the sequence of MT-2 and that of native ubiquitin. The sequence of ubiquitin was further confirmed and supported by amino acid and parital sequence anlysis of fragments obtained by digestion of maleated ubiquitin with chymotrypsin or staphylococcal protease.

Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins.

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Cells of several strains of Streptococcus gordonii attached in much higher numbers to experimental pellicles formed from samples of submandibular or parotid saliva on hydroxyapatite (HA) beads than to buffer controls. The nature of the salivary components responsible were investigated by preparing experimental pellicles from chromatographic fractions of submandibular saliva obtained from Trisacryl GF 2000M columns. Adhesion of S. gordonii Blackburn was promoted by two groups of fractions. The adhesion-promoting activity in the first group of fractions was associated with the family of acidic proline-rich proteins (PRPs), while that of the second group is as yet unidentified. Experimental pellicles prepared by treating HA with 2 micrograms of pure 150-amino-acid-residue PRPs (PRP-1, PRP-2, and PIF-s) promoted adhesion of S. gordonii Blackburn cells to an extent comparable to that obtained with unfractionated saliva. However, pellicles prepared from a 106-residue PRP (PRP-3) were significantly less effective, and those prepared from the amino-terminal tryptic peptide (residues 1 to 30) of the PRP and the salivary phosphoprotein statherin were completely ineffective in promoting adhesion. Although adhesion of several strains of S. gordonii was promoted by adsorbed PRP-1, the adhesion of several strains of Streptococcus sanguis or Streptococcus oralis was either not affected or only weakly enhanced by this protein. S. gordonii cells bound avidly to PRPs adsorbed onto HA beads, but the streptococci did not appear to bind PRPs in solution, since concentrations of PRP as high as 200 micrograms/ml did not inhibit binding of bacterial cells to pellicles prepared from pure PRP. S. gordonii cells also attached well to PRP or a synthetic decapeptide representing residues 142 to 150 of the PRP when the peptide was linked to agarose beads. Studies with a series of synthetic decapeptides indicated that the minimal segment of PRP which promoted high levels of S. gordonii adhesion was the carboxy-terminal dipeptide Pro-Gln (residues 149 and 150).

Human salivary acidic proline-rich proteins (PRPs) constitute a significant fraction of the total salivary protein and possess important biological activities. Different genetic and post-translationally processed forms of the PRPs exhibit significant quantitative variations in several of these activities, especially the modulation of salivary calcium phosphate chemistry and oral bacterial adhesion. To quantify and understand these differences, we have developed a high-performance liquid chromatography (HPLC) method to identify and measure individual PRPs in saliva. The data obtained permit the identification of PRP polymorphisms and phenotypes, the determination of the relative amounts of PRPs derived from the two loci, PRH1 and PRH2, and the measurement of the extent of post-translational cleavage of the primary polypeptide products. Substantial inter-gland and inter-individual variations were found in relative amounts of PRPs derived from the two loci (at least two-fold), and in post-translational cleavage (greater than two-fold), both of which are likely to be biologically significant. Also in this study, the presence of what appear to be minor amounts of numerous variant PRPs in glandular secretions was observed, and two uncommon PRP polymorphisms were identified in the 127 subjects studied.

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