Recognition of substrates by tyrosylprotein sulfotransferase. Determination of affinity by acidic amino acids near the target sites.

The sulfation of proteins by tyrosylprotein sulfotransferase (TPST) is highly site-specific. In this study, we examined the sequence specificity of the target site for TPST by determining the kinetics of rat liver TPST with peptides related to the sequence of the C4 component of complement. The data obtained from this study demonstrate that selective elimination of negative charges from the -5 to +5 region of the acceptor tyrosine, either by removal or by isosteric substitution or the acidic amino acids in the region, produced a substantial change in the Km value, with relatively little effect on Vmax. Substitutions at -1 and +1 positions increase the Km value by 22- and 4-fold, respectively, whereas removal of the acidic amino acids from the -5, -4, or +4 positions increased the Km values by a factor of 2-4. The effect of elimination of an acidic amino acid on the Km value was constant and specific for its particular position in relation to tyrosine, and the effect of modification of more than one amino acid was multiplicative. This study provides evidence that: 1) acidic residues near tyrosines promote sulfation by increasing the affinity of enzyme-substrate binding and have little effect on catalytic rate; 2) the contribution of each acidic residue to affinity for TPST is independent and varies according to position relative to the acceptor tyrosine; and 3) the enzyme interacts with a segment of at least 4-5 residues on each side of the tyrosine, with the residues on the -1 and +1 positions being the most important determinants. In general, residues on the NH2-terminal side of the tyrosine have a greater effect on affinity for TPST.     

Inhibitors of the sulfation of proteins, glycoproteins, and proteoglycans

Two categories of compounds, substrates of sulfation and sulfate analogs, were tested for the ability to inhibit sulfation of macromolecules secreted by HepG2 cells. Several compounds which most effectively inhibited sulfation without toxic effects on cells were tested for their relative inhibition of sulfation of tyrosine residues (using the fourth component of complement as a model substrate), of N-linked oligosaccharides (alpha 2HS-glycoprotein as substrate), and of proteoglycans. Inhibitors decreased the sulfation of all three classes of substrate, but not always equally. Use of inhibitors from both categories in combination yielded synergistic effects, with more effective inhibition of sulfation and low toxicity. Such combinations of inhibitors should provide a valuable tool for probing the significance of the sulfation of macromolecules.     

Improved detection of intact tyrosine sulfate-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in linear negative ion mode

Sulfation of tyrosine residues is a common post-translational modification, but detecting and quantitating this modification poses challenges due to lability of the sulfate group. The goal of our studies was to determine how best to detect and to assess the stoichiometry of this modification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Sulfated and nonsulfated forms of peptides—hirudin(55–65), caerulein, and cholecystokinin octapeptide and phosphorylated and nonphosphorylated pp60-c-src (521–533)—were analyzed using several matrices: sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DBA), and cyano-4-hydroxycinnamic acid (CHCA). Intact sulfated peptides were difficult to detect using positive ion mode; peptides were observed as desulfated ions. Phosphorylated peptide was stable and was detected in positive and negative ion modes. Detection of sulfated peptides improved with: (1) Analysis in negative ion mode, (2) Decreased laser power, (3) Matrix selection: DBA ≥ SA > CHCA. In negative ion mode, desorption/ionization of sulfated peptide was equivalent or more efficient than nonsulfated peptide, depending on conditions of analysis. Examination of a tryptic digest of α₂-antiplasmin detected the single site of sulfation in negative ion mode but not in positive ion mode. We conclude that improved detection of sulfated peptides can be achieved in negative ion mode. Dual analysis in positive and negative ion modes serves as a potential means of identifying peptides with labile modifications such as sulfation and distinguishing them from phosphorylation.     

Diverse range of small peptides associated with high-density lipoprotein

High-density lipoproteins (HDL) were examined as potential carriers of small peptides in plasma. HDL purified by density gradient centrifugation was delipidated and fractionated by size-exclusion chromatography under denaturing conditions. By HPLC and mass spectrometry, more than 100 peptide components were found in the size range from 1000 to 5000 Da. By sequence analysis, peptides were identified as fragments of proteins such as apolipoproteins, fibrinogen, alpha1-proteinase inhibitor, and transthyretin. The results indicate that purified HDL bears a complex range of small peptides. It is unclear whether the peptides have any significant functional role as apolipopeptides, but they may represent a pathway for peptide delivery or scavenging and a significant reservoir of plasma peptides for diagnostic evaluation.     

Identification of two sites of sulfation of human heparin cofactor II

Tyrosine sulfate was identified as a constituent of human heparin cofactor II by analysis of sulfate-labeled protein secreted by a human hepatoma-derived cell line and of purified protein from human plasma. Alkaline hydrolysis of heparin cofactor II released tyrosine sulfate as demonstrated by anion-exchange high performance liquid chromatography of hydrolysates. Two sites of sulfation were identified, and the amino acid sequences of the sites were established by sequential Edman degradation of sulfate-containing tryptic peptides that were isolated by reverse-phase high performance liquid chromatography. Each peptide contains only a single tyrosine residue so that the sites of sulfation can be assigned unambiguously. The two sites of sulfation are separated by 13 residues and represent an internal sequence repeat in the heparin cofactor II molecule. The two sites have the following sequences. Glu56-Asp-Asp-Asp-Tyr(SO4)-Leu-Asp62 Glu69-Asp-Asp-Asp-Tyr(SO4)-Ile-Asp75 Sulfate-labeled heparin cofactor II formed a covalent complex with thrombin in a heparin-dependent manner. Thus, the sulfate-containing form of the protein was shown to be biologically active. The characteristic sulfate-containing segment of heparin cofactor II, which contains 17 acidic amino acid residues over a span of 30 residues, may contribute to the unique properties of this thrombin inhibitor.     

Sulfation of a tyrosine residue in the plasmin-binding domain of alpha 2-antiplasmin

Sulfation of human alpha 2-antiplasmin, the major plasma inhibitor of fibrinolysis, was examined using both protein isolated from human plasma and protein synthesized and biosynthetically labeled with [35S]sulfate by a human hepatoma-derived cell line. Linkage of sulfate to tyrosine was demonstrated by recovery of labeled tyrosine sulfate after base hydrolysis of sulfate-labeled alpha 2-antiplasmin. Analysis by reverse-phase high performance liquid chromatography of peptides released from alpha 2-antiplasmin by cleavage with trypsin or cyanogen bromide indicated that sulfate is linked to a single segment of the protein. A cyanogen bromide peptide corresponding to the sulfate-labeled peptide was prepared from alpha 2-antiplasmin isolated from human plasma. Consistent with the presence of tyrosine sulfate in this peptide, its chromatographic elution was altered by treatment with acid under conditions which release sulfate from a tyrosine residue. No peptide in the total digest of alpha 2-antiplasmin by cyanogen bromide eluted at the position of the peptide following desulfation, suggesting that all of the protein is in a sulfated form. The sequence of the sulfate-containing cyanogen bromide peptide as determined by sequential Edman degradation, amino acid composition, and fast atom-bombardment-mass spectrometry was: Glu-Glu-Asp-Tyr(SO4)-Pro-Gln-Phe-Gly-Ser-Pro-Lys-COOH. This peptide is a segment of the previously identified plasmin-binding domain of alpha 2-antiplasmin.     

Allosteric changes in thrombin's activity produced by peptides corresponding to segments of natural inhibitors and substrates.

Acidic synthetic peptides corresponding to segments of several nonhomologous proteins (hirudin, residues 54-65; heparin cofactor II, residues 54-75; and fibrinogen, residues 410-427 of the gamma B-chain) inhibit thrombin's cleavage of fibrinogen without blocking the enzyme's active site. Here, we examined effects of these peptides on thrombin's cleavage of protein C and small peptides. Activation of protein C by thrombin in the absence of calcium was inhibited by all of the peptides. Maximal inhibition was 60%, and no greater inhibition was produced by higher peptide concentrations. This differed from progressive inhibition of protein C activation by increasing peptide concentrations in the presence of thrombomodulin and calcium. Potencies of the peptides were in the order hirudin-(54-65) greater than heparin cofactor II-(54-75) greater than gamma B-chain-(410-427). Sulfation of the tyrosine residue in hirudin-(54-65) increased its potency about 10-fold, similar to changes in anticlotting activity. The peptides were activators rather than inhibitors of the cleavage of small chromogenic substrates. In the presence of the peptides, the affinity of thrombin for the substrates S-2366 (pyro-Glu-Pro-Arg-4-nitroanilide), Chromozyme TH (tosyl-Gly-Pro-Arg-4-nitroanilide), and S-2251 (D-Val-Leu-Lys-4-nitroanilide) increased 1.5-2-fold with little change in the Vmax of substrate cleavage. Potencies of peptides in these allosteric effects on thrombin was in the same order as for their other effects. The similar actions of these nonhomologous peptides, which are believed to bind to thrombin's anion-binding exosite, suggest that binding of any peptide to this site exerts the same allosteric effect on thrombin's active site. Interactions of these peptides with thrombin may serve as models for regulation of thrombin's interactions with natural substrates and inhibitors.     

The dynamic range problem in the analysis of the plasma proteome

One of the greatest challenges in analyzing the plasma proteome is the wide range of concentration of different proteins. The current study examines the range of protein concentration for 18 proteins measured over a year in a clinical laboratory to provide data on pathological extremes in protein concentrations. The complete measured range, from upper limits for albumin to lowest values for thyroid-stimulating hormone (TSH), represented more than 10 logs of molar abundance. A number of plasma proteins measured in the clinical laboratory varied over a concentration range spanning more than 4 logs, and limits of detection of clinical assays were inadequate to assess full concentration ranges of several proteins. Considering reported values from studies using higher sensitivity assays suggest that plasma concentrations of prostate-specific antigen (PSA), human chorionic gonadotropin (hCG), and cardiac troponin I vary by more than 7 logs. All of the plasma proteins measured in the present study represent secretory proteins or highly expressed components of specific tissues. Thus, the dynamic range for these components is likely to greatly underestimate the total range of protein concentration in the plasma proteome.     

Mass determination of major plasma proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) serves as a rapid and accurate means to determine masses of proteins independent of their shapes or interactions with other molecules. It provides one of the most fundamental characterizations of major plasma proteins. Purified proteins in saline or serum specimens were prepared for analysis by dilution, mixing with a solution of sinapinic acid, and drying on a target plate. Specimens were analyzed in a linear TOF mode with external calibration. Analyses of 24 purified plasma proteins showed predominance of singly charged ions with lesser amounts of dimer and doubly charged monomer, and provided measured masses for these proteins. A number of proteins, including albumin, transferrin, apolipoproteins A-I, A-II, C-I, C-II, and C-III, and prealbumin, could be analyzed directly in serum with appropriate dilution. Measured values for masses of major plasma proteins will assist in analysis of serum and plasma. It is possible to analyze a number of components by MALDI-TOF/MS directly in diluted serum. Extremely simple sample preparation techniques may be useful in analyzing structural variation of several major plasma proteins, particularly those with masses <30 kDa, including a number of apolipoproteins and markers of nutritional status or acute phase responses.     

Preparation of soluble peptide libraries: application to studies of platelet adhesion sequences.

We describe chemical synthesis of peptide mixtures that equally express many sequence combinations. Using 65 couplings of single amino acids, five mixtures were prepared with the sequences Tyr-Gly-Arg-Gly-Yyy-Xxx-Xxx, where Yyy is Ser, Asp, Arg, Asn, or Glu, and Xxx is any amino acids. Compositional and sequence analyses supported full representation of all amino acids, except isoleucine was deficient in the sixth position. The data suggest formation of a repertoire of 1,900 sequence combinations (5 x 19 x 20). The mixture with Asp as the fifth residue inhibited platelet adhesion to fibronectin more effectively than the other mixtures. Peptide libraries offer a new tool for investigating bioactive peptides.