Phosphopeptides derived from human salivary acidic proline-rich proteins. Biological activities and concentration in saliva.

Human saliva contains a large number of phosphopeptides derived by cleavage of acidic proline-rich proteins (APRPs). These peptides were purified by column chromatography and they constituted 0.5% of APRPs in parotid saliva, but 11% of APRPs in saliva expectorated from the mouth (whole saliva), indicating that there is considerable cleavage of APRPs after secretion from the gland. Similarly to APRP, the phosphopeptides bind Ca2+, but they accounted for only 4% of protein-bound Ca2+ in whole saliva. APRPs as well as the phosphopeptides inhibited formation of hydroxyapatite, but, whereas 19-20 micrograms of APRP was needed for 50% inhibition, only 0.7-3.3 micrograms of purified peptides was needed for the same degree of activity, and the phosphopeptides accounted for 18% of total inhibitory activity in whole saliva. All phosphopeptides adsorbed on hydroxyapatite in vitro, and adsorption of phosphopeptides on tooth surfaces in vivo could also be demonstrated, indicating that they would be able to inhibit unwanted mineral formation on the tooth surface in vivo. Degradation of APRPs after secretion therefore does not lead to a loss of their biological activities.     

Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.     

The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins.

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.     

Salivary peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40-50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400-600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln-Gly cleavages are largely associated with PRP classes, while Tyr-Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sj?gren's syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.     

Salivary peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40–50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400–600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln–Gly cleavages are largely associated with PRP classes, while Tyr–Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sjögren’s syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.     

Proteolytic enzymes of Neurospora crassa. Purification and some properties of five intracellular proteinases.

Five intracellular proteolytic enzymes from Neurospora crassa were isolated and partially characterized: an acidic and an alkaline endopeptidase, one carboxypeptidase and two aminopeptidases. All these proteinases were purified from the same crude extract to homogenity by heat treatment, precipitation with ammonium sulfate, chromatography on DEAE-cellulose, CM-cellulose, DEAE-Sephadex, hydroxyapatite and by gel filtration. The acid proteinase hydrolysed acid-denatured haemoglobin at pH 3.0. The alkaline proteinase and the carboxypeptidase are serine proteinases that require a sulfhydryl group for activity. The aminopeptidases are both metallo-proteinases; one posseses broad specifity to the B-chain of oxidized insulin, the other posseses only narrow specifity and can only split the N-terminal basic amino acids of peptides.     

Salivary proline-rich proteins

Summary Proline-rich proteins are major components of parotid and submandibular saliva in humans as well as other animals. They can be divided into acidic, basic and glycosylated proteins. The primary structure of the acidic proline-rich proteins is unique and shows that the proteins do not belong to any known family of proteins. The proline-rich proteins are apparently synthesized in the acinar cells of the salivary glands and their phenotypic expression is under complex genetic control.The acidic proline-rich proteins will bind calcium with a strength which indicates that they may be important in maintaining the concentration of ionic calcium in saliva. Moreover they can inhibit formation of hydroxyapatite, whereby growth of hydroxyapatite crystals on the tooth surface in vivo may be avoided. Both of these activities as well as the binding site for hydroxyapatite are located in the N-terminal proline-poor part of the protein. Little is known about the functions of the glycosylated and basic proline-rich proteins.     

Localization of the phosphorylations of polyomavirus large T antigen.

Polyomavirus large T antigen is phosphorylated on both serine and threonine residues at a ratio of approximately 6 to 1. This phosphorylation could be resolved into a series of nine Staphylococcus aureus V8 phosphopeptides. All of these were found in an N-terminal chymotryptic fragment with a molecular weight of 57,000. A C-terminal formic acid fragment of 50,000-molecular-weight lacked phosphate. Therefore, unlike simian virus 40 large T antigen, polyomavirus large T antigen has no significant C-terminal phosphorylation. Limited V8 and hydroxylamine cleavage showed that the phosphorylations can be localized to two different portions of the molecule. A significant fraction of the phosphate was localized in the N-terminal portion of the molecule before residue 183. Within this region V8 peptides 4, 8, and 9 represented phosphorylations that were more proximal, while peptides 1, 2, and 3 included more distal phosphorylations. None of these phosphorylations appeared analogous to those of simian virus 40 large T antigen. V8 phosphopeptides 5 and 7 were more distal and could be distinguished in biological experiments from the N-terminal phosphorylations. Formic acid mapping suggested that much, if not all, of this phosphorylation is located between residues 257 and 285.     

Hormone-dependent phosphorylation of the avian progesterone receptor

Progesterone receptors are phosphoproteins, in which phosphorylation has been proposed as a control mechanism for some stages of hormone action. Progesterone administration was shown to increase phosphorylation of the receptor from both cytosol and nuclear extracts of whole cells. We have analyzed the receptor phosphopeptides generated by chemical and proteolytic cleavage to assess the number of phosphorylation sites and their approximate location in the receptor. Progesterone receptor was labeled in situ in the presence or absence of hormone in medium containing [32P] orthophosphate, isolated by immunoprecipitation, and then digested with several proteases. The resulting 32P-labeled peptides were resolved by either two-dimensional electrophoresis:chromatography or by reverse-phase high performance liquid chromatography. Multiple phosphopeptides (3-6) were detected after cleavage with trypsin, chymotrypsin, or V8 protease. Major increases in phosphorylation occurred at existing sites since after hormone treatment no new phosphopeptides were found. Individual phosphopeptides showed variable increases in phosphorylation of 1.5-5-fold. The A and B receptor forms showed identical phosphorylation patterns, indicating similar processing in vivo. The phosphopeptide pattern for receptor in nuclear extracts resembled that of cytosol receptor. Chemical cleavage was used to assess the distribution of phosphorylation sites. Cyanogen bromide produced a large 40-kDa polypeptide which contained all of the phosphorylation sites and comprised the residues 129-449. Hydroxylamine was used to cleave a unique bond, Asn-372-Gly-373, in the 40-kDa polypeptide. All of the phosphorylation sites were located on the amino-terminal side of the cleavage. Thus, all of the phosphorylation sites were localized to a specific region (Met-129 to Asn-372) of the progesterone receptor that does not include either the DNA or steroid binding domains.     

The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution

The aim of this study was the development of a method based on the coupling of RP-HPLC and ESI-MS for identifying and quantifying proteins and peptides secreted by human salivary glands in vitro. Salivary gland specimens, obtained from informed patients undergoing surgical resection, were incubated in an optimized medium. Incubation media of glandular specimens, selected on the basis of cytomorphological and ultrastructural analysis, were investigated by HPLC-MS. Several salivary peptides/proteins, previously recognized in human whole saliva, were searched for along the chromatogram by the selected ion monitoring (SIM) strategy. Analysis of the incubation media of parotid glands revealed the presence of basic PRPs PC, PD, PH, IB-1, II-2, and acidic PRP-1 and PRP-3 in all of the investigated samples. Basic PRPs PB and PA, acidic PRPs, and cystatins SN and S1 were detected in all of the incubation media of submandibular glands, whereas histatin 1 was detected in only one sample. Moreover, the method allowed detection of some post-translational derivatives of known salivary proteins, as well as of several previously unidentified small peptides. The present method represents a sensitive and powerful instrument to detect peptides and proteins secreted by human salivary glands in vitro.     

羧肽酶Y标记蛋白质羧基端方法的优化及应用

蛋白质水解是一种重要的翻译后修饰,它在许多生化过程 (如细胞凋亡和肿瘤细胞转移等) 中起着极其重要的作用。鉴定蛋白质水解位点可以进一步加深我们对这些生化过程的认识。尽管蛋白质氨基端标记方法和蛋白质组学在复杂生物体系中鉴定获得了许多蛋白质的水解位点,但这种方法存在固有的缺陷。羧基端标记方法是另一种可行的鉴定蛋白质水解位点的方法。本文优化了蛋白质羧基端生物酶标记方法,提高了亲和标记效率,从而可以更好地利用正向分离方法对蛋白质羧基端多肽进行分离并用质谱鉴定。我们用优化后的羧基端标记方法来标记大肠杆菌Escherichia coli复杂蛋白样品后鉴定到了120多个蛋白质羧基端多肽和内切多肽。在其所鉴定的蛋白质水解位点中,我们发现了许多已知和未知的位点,这些新的水解位点有可能在正常生化过程的调控发挥着重要的作用。该研究提供了一个可以与蛋白质氨基端组学互为补充、可在复杂体系中鉴定蛋白质水解的方法。     

Unbiased Selective Isolation of Protein N-terminal Peptides from Complex Proteome Samples Using Phospho Tagging (PTAG) and TiO2-based Depletion

A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products.     

Synthesis of a potent photoreactive acidic γ-secretase modulator for target identification in cells

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimer´s disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Compounds named γ-secretase modulators (GSMs) can shift the substrate cleavage specificity of γ-secretase toward the production of non-amyloidogenic, shorter Aβ fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of γ-secretase), supporting a mode of action involving binding to γ-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in γ-secretase proteolytic specificity should pave the way for the development of improved drugs against AD.     

Identification of proteolytic cleavage sites by quantitative proteomics

The identification of natural substrates and their cleavage sites is pivotal to defining proteolytic pathways. Here we report a novel strategy for the identification of the signature of proteolytic cleavage events based on quantitative proteomics. Lysine residues in proteins are blocked by guanidination so that free N-terminals can be labeled with amine-specific iTRAQ reagents. The quantitative nature of iTRAQ reagents allows us to distinguish N-terminals newly formed by proteolytic treatment (neoepitopes) from original N-terminals in proteins. Proteins are digested with trypsin and analyzed using MALDI-TOF/TOF mass spectrometry. Peptides labeled with iTRAQ reagents are distinguished from other peptides by exhibiting intense signature ions in tandem mass spectrometry analysis. A corresponding data acquisition strategy was developed to specifically analyze iTRAQ tagged N-terminal peptides. To validate the procedure, we examined a set of recombinant Escherichia coli proteins that have predicted caspase-3 cleavage motifs. The protein mixture was treated with active or inactive caspase-3 and subsequently labeled with two different iTRAQ reagents. Mass spectrometric analysis located 10 cleavage sites, all corresponding to caspase-3 consensus. Spiking caspase-cleaved substrate into a human cell lysate demonstrated the high sensitivity of the procedure. Moreover, we were able to identify proteolytic cleavage products associated with the induction of cell-free apoptosis. Together, these data reveal a novel application for iTRAQ technology for the detection of proteolytic substrates.     

Metal oxide-based enrichment combined with gas-phase ion-electron reactions for improved mass spectrometric characterization of protein phosphorylation

Gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), are advantageous for characterization of protein posttranslational modifications (PTMs), because labile modifications are not lost during the fragmentation process. However, at least two positive charges and relatively abundant precursor ions are required for ECD due to charge reduction and lower fragmentation efficiency compared to conventional gas-phase fragmentation techniques. Both these criteria are difficult to fulfill for phosphopeptides due to their acidic character. The negative ion mode operation of EDD is more compatible with phosphopeptide ionization, but EDD suffers from a fragmentation efficiency even lower than that of ECD. Recently, metal oxides such as ZrO 2 and TiO 2 have been shown to provide selective enrichment of phosphopeptides from proteolytic digests. Here, we utilize this enrichment strategy to improve ECD and EDD of phosphopeptides. This approach allowed determination of the locations of phosphorylation sites in highly acidic, multiply phosphorylated peptides from complex peptide mixtures by ECD. For singly phosphorylated peptides, EDD provided complementary sequence information compared to ECD.     

Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme

The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals

Osteonectin, an acidic noncollagenous protein of bone and dentin, has affinity to hydroxyapatite crystals. Binding sites to hydroxyapatite of this protein were determined by a proteolytic experiment and an in vitro binding experiment using synthetic peptide analogues. Osteonectin was adsorbed on hydroxyapatite crystals and digested with trypsin. A peptide was left adsorbed on the crystal even after the digestion. The peptide was identified as an amino terminal peptide containing glutamic acid-rich sequences, which have been assumed to be possible hydroxyapatite-binding sites. Poly glutamic acid sequences were synthesized as models of the binding sites. Glu₆ peptide was bound to the hydroxyapatite with a dissociation constant of 2.4 μM. Peptides containing fewer glutamic acids had lower affinity to the crystal. Effects of these peptides on in vitro mineralization were examined by a gel system in microtiter plates. The Glu₆ peptide had a positive effect on the mineralization in this system, whereas Asp₆ peptide had a negative effect. These effects indicate the presence of an interaction between these peptides and mineral crystals.