The presence and origin of phosphopeptides in human saliva.

The presence of phosphopeptides in whole saliva (saliva expectorated from the mouth) was demonstrated and their origin was evaluated. Whole saliva contained much larger numbers of small phosphopeptides than are found in the glandular secretions. Most of these originated from the acidic proline-rich proteins (PRPs) in the major salivary glands and were formed, after secretion into the oral cavity, as a result of rapid degradation by proteolytic enzymes from extraglandular sources contained in sediment from whole saliva. Some peptides may have been formed by cleavage of basic PRPs, but other phosphoproteins apparently contributed little to the observed phosphopeptides. Most of the enzymes that produced phosphopeptides are serine proteinases. The gel-electrophoretic band patterns of the phosphopeptides obtained from 26 individuals of various acidic-PRP phenotypes were remarkably similar, demonstrating that the enzymes responsible were generally present in the population surveyed and that similar cleavages occur regardless of the nature of the acidic PRPs. Many of these peptides were N-terminal proteolytic cleavage products of acidic PRPs. The N-terminal phosphorylated region of acidic PRPs contains various biological activities, such as inhibition of hydroxyapatite formation, calcium binding and binding to hydroxyapatite, the major mineral of teeth. The demonstration of these phosphopeptides in the saliva that is in contact with the oral surface may therefore be of biological importance.     

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, ¹⁸O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes^1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of ¹⁸O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao⁴, Miyagi and Rao⁵ and Ye et al.⁶). ¹⁸O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques⁷. Depending on the protease utilized, ¹⁸O-labeling can result in the incorporation of up to two ¹⁸O-atoms in the C-terminal carboxyl group of the cleavage product³. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction⁸. In our PALeO (protease-assisted labeling employing ¹⁸O-enriched water) adaptation of enzymatic ¹⁸O-labeling, we utilized 50% ¹⁸O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases⁹ and monitor proteolytic reactions^10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second ¹⁸O-atom. Such "double-labeling" enzymes can be used for postdigestion ¹⁸O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing ¹⁸O-enriched water beyond enzymes and uses acidic pH conditions to introduce ¹⁸O-stable isotope signatures into peptides.     

Profiling protease activities by dynamic proteomics workflows

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.     

Proteinases and their inhibitors in cells and tissues

A large body of evidence has been assembled to indicate the substantial importance of proteolytic processes in various physiological functions. It has recently become clear too that endo-acting peptide bond hydrolases provisionally characterized and classified at present as serine, cysteine, aspartic and metallo together with unknown catalytic mechanism proteinases sometimes act in cascades. They are controlled by natural proteinase inhibitors present in cells and body fluids. In the first part of the present monograph the author was concerned to present an overview on the morphological and physiological approach to localization, surveying reaction principles and methods suitable for visualization of proteolytic enzymes and their natural and synthetic inhibitors. In the second part the roles played by proteinases have been summarized from the point of view of cell biology. The selection of earlier and recent data reviewed on the involvement of proteolysis in the behavior of individual cells reveals that enzymes, whether they be exogeneous or intrinsic, can be effective and sensitive modulators of cellular growth and morphology. There exists a close correlation between malignant growth and degradation of cells. It appears likely that as yet unknown or at least so far inadequately characterized factors that influence the survival or the death of cells may turn out to be proteinases. The causal role of extracellular proteolysis in cancer cell metastases, in stopping cancer cell growth and in cytolysis remains for further investigated. Ovulation, fertilization and implantation are basic biological functions in which proteolytic enzymes play a key role. The emergence of new approaches in reproductive biology and a growing factual basis will inevitably necessitate a reevaluation of present knowledge of proteolytic processes involved. The molecular aspects of intracellular protein catabolism have been discussed in terms of the inhibition of lysosomal and/or non-lysosomal protein breakdown. Peptide and protein hormone biosynthesis and inactivation are still at the centre of interest in cell biology, and a number of proteinases have been implicated in both processes. A number of conjectures partly based on the author's own work have been discussed which suggest the possibility of the involvement of proteolysis in exocytosis and endocytosis. The author's optimistic conclusion is that through the common action of biochemists, cell biologists, cytochemists, and pharmacologists the mystery of cellular proteolysis is beginning to be solved.     

Aspartic proteinases in the digestive tract of marine decapod crustaceans

Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.     

Thiol peptide hydrolases from animal tissues, their structure and function

Data on properties, structure and biological functions of a variety of thiol (cysteine) peptide hydrolases from animal tissues have been summarized. This large group of diverse intracellular enzymes involves both endo- and exopeptidases. Best studied are lysosomal thiol peptide hydrolases: cathepsins B, H and L, the primary structure of which is deciphered. They present a family of homologous proteins, structurally similar to papain. Ca2+-dependent neutral proteinases is another family of related proteins. The biological functions of various thiol peptide hydrolases are considered: their participation in protein turnover, post-translational processing, regulation of unidirectional biological processes and metabolic refolding. Data on endogenous inhibitors of thiol peptide hydrolases and on regulation of enzymic activity are presented.     

New proteolytic enzymes in yeast

Yeast mutants lacking three proteolytic enzymes—proteinase B, carboxypeptidase Y, and carboxypeptidase S—have been constructed. Search for new proteolytic activities in these mutants with the aid of chromogenic peptide substrates developed for serum proteinases led to the detection of new proteolytic activities, active in the neutral pH range. Sephadex chromatography of a 100,000g supernate of mutant extracts, tests against four different substrates, and partial characterization of their sensitivity to various inhibitors indicate multiple activities. Two activities, called proteinase M and proteinase P, were found in the sedimentable membranous fraction of mutant extracts.     

Cysteine peptidases and their inhibitors in breast and genital cancer

Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.     

An unusual specificity in the activation of neutrophil serine proteinase zymogens

The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusual C-terminal and N-terminal peptide extensions absent from the mature proteins. We have used biosynthetic radiolabeling and radiosequencing techniques to identify the kinetics of activation of both proteinases in the promonocyte-like cell line U937. We find that both N- and C-terminal extensions are removed about 90 min after the onset of synthesis, resulting in the activation of the proteinases. HNE and cat G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Activation results from cleavage following a glutamic acid residue to give an activation specificity opposite to those of almost all other serine proteinase zymogens, but shared, possibly, by the "granzyme" group of related serine proteinases present in the killer granules of cytotoxic T-lymphocytes and rat mast cell proteinase II.     

Proteolysis in eukaryotic cells. Identification of multiple proteolytic enzymes in yeast

A previous study led to the discovery of new proteinases in yeast (Achstetter, T., Ehmann, C., and Wolf, D. H. (1981) Arch. Biochem. Biophys. 207, 445-454). The search for proteolytic enzymes active in the neutral pH range has been extended. Studies were done on a mutant lacking four well-known proteinases involved in protein degradation, the two endoproteinases A and B and the two carboxypeptidases Y and S. Twenty-nine chromogenic peptides (amino terminally blocked peptidyl-4-nitroanilides) as well as [3H]methylcasein were used as substrates in this search. For the detection of endoproteolytic activity using chromogenic peptide substrates two versions of the assay were used. In one system the direct cleavage of the 4-nitroanilide bond was measured. In the second, the cleavage of the chromogenic peptide at some site other than the 4-nitroanilide bond was measured. Both variations led to the discovery of multiple proteinase activities. Regulation of these proteolytic activities under different growth conditions of cells was observed. Proteolytic activity on [3H]methylcasein was also found. Ion-exchange chromatography and gel filtration were used for the reproducible separation of the multiple proteolytic activities.     

Preliminary analysis of the proteolytic enzymes in the excretory-secretory products of the adult stages of the dog hookworm Uncinaria stenocephala

The paper describes an introductory characterisation of proteinases present in the excretory-secretory products (ESP) of adult Uncinaria stenocephala. In SDS-PAGE gelatine substrate gels ESP resolved as a six bands of proteolytic activity, with a molecular weight of 182, 159, 98, 50, 39 and 26 kDa. The 98 and 39 kDa components were serine proteinases. The 50 kDa band was sensitive to a metalloproteinase inhibitor. The 26 kDa component was highly sensitive to cysteine proteinase inhibitors and was also partially inhibited in the presence of EDTA. The bands of 182 and 159 kDa were sensitive to a Zn-metalloproteinase inhibitor. The enzymes present in ESP showed the highest proteolytic activity at pH 8-9. Quantitative analysis revealed maximum proteolytic activity of the polypeptides of 159 and 182 kDa at pH 7; 98 and 26 kDa at pH 8 while the 50 kDa and 39 kDa components showed the highest activity at pH 9.     

Assignment of endogenous substrates to enzymes by global metabolite profiling

Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.     

A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases of antigen-presenting cells causes a profound inhibition of both the proteolytic degradation and the presentation of the synthetic antigen dinitrophenyl-poly-L-lysine. In contrast, the presentation of another synthetic antigen, the copolymer of L-glutamic acid and L-alanine, was enhanced by the same inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing.     

SELDI-TOF-MS of saliva: methodology and pre-treatment effects

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.     

Lipid species profiling: a high-throughput approach to identify lipid compositional changes and determine the function of genes involved in lipid metabolism and signaling

The development of electrospray ionization mass spectrometry has provided the foundation for the development of strategies to identify and quantify complex lipids from unfractionated extracts of small biological samples. In the 1990s, the feasibility of detailed lipid profiling was demonstrated; in the past two years, analytical strategies have been extended to include classes of lipids that are unique to plants. High-throughput lipid profiling by electrospray ionization tandem mass spectrometry, in combination with forward- or reverse-genetics approaches, has recently been utilized to identify lipid metabolic pathways that are involved in plant development and stress responses, to specify the roles of particular genes and enzymes in plant responses to environmental cues, to determine the lipid species that serve as the substrates and products of specific enzymes, and to identify lipid-metabolizing enzymes that are involved in varied plant processes.     

Strongyloides ransomi: proteolytic enzymes from larvae

The filariform larvae of Strongyloides ransomi can infect their hosts by penetration through skin. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Homogenates prepared in 0.2 M citrate, pH 4.0, contain two thiol-dependent proteinases with molecular weights of approximately 32,000 and 28,000. These proteinases have an acidic pH optimum and show substrate preferences and inhibitor susceptibilities similar to the vertebrate acidic cysteinyl proteinases. Homogenates prepared in 0.1 M Tris, pH 7.5, contain multiple proteolytic enzymes, active against both Azocoll and synthetic substrates. These enzymes do not require thiols for activity and they have an alkaline pH optimum. The enzymes are inhibited by both chelating agents and heavy metals, but not by serine-proteinase inhibitors. Extracts prepared in 0.1 M Tris-HCl, pH 7.5, contain endogenous proteinase inhibitors.     

Species comparison of renal proteolytic activity for human myelin basic protein peptide 43-88

1. A species comparison was conducted on the proteolytic activity in human, dog, rabbit, guinea-pig and rat kidney which can degrade human myelin basic protein peptide 43-88. 2. In rat kidney the degrading activity occurred over a pH range of 4-11.5 with the greatest activities at pH 5 and 9. The peptide degrading activity in human, dog, rabbit and guinea-pig kidney was considerably less than in the rat and occurred predominantly at pH 7 with lesser activity at pH 9. 3. The effects of inhibitors of proteolytic enzymes indicated that the peptide degrading activities at the same two pH's of dog, rabbit and guinea-pig were similar to one another but differed from that of human. 4. These results indicate that the activity for degrading a potential autoantigenic material is widespread in renal tissue among different species and that different enzymes are involved. More generally, these findings suggest that renal proteinases differ among commonly used laboratory animals and also differ from the human enzymes.     

鱼糜凝胶劣化现象相关蛋白水解酶

鱼糜凝胶劣化现象发生在鱼糜凝胶化过程中,导致鱼糜制品凝胶弹性下降,品质降低。研究表明,引起鱼糜凝胶劣化现象的原因是鱼糜内含的蛋白水解酶的作用。与该现象相关的蛋白水解酶类有肌浆型和肌原纤维结合型之分。大部分肌浆型蛋白水解酶为水溶性的,可以在鱼糜制备过程中漂洗去除。肌原纤维结合型蛋白酶不能通过漂洗有效去除,是引发鱼糜凝胶劣化现象的重要相关酶类。已证实的凝胶劣化相关肌原纤维结合型酶类包括丝氨酸型蛋白酶和溶酶体型组织蛋白酶。     

The proteolytic activities of chymopapain, papain, and papaya proteinase III

The three proteinases present in papaya latex: papain (EC 3.4.22.2) chymopapain and papaya proteinase III (EC 3.4.22.6), were standardized by active-site titration, and compared in proteolytic activity against azocasein, serum albumin and cartilage proteoglycan. The activities were all of the same order, although there were differences in pH dependence. SDS-polyacrylamide gel electrophoresis of the early products of digestion of albumin and phosphorylase a showed very similar patterns for the three papaya proteinases. Kinetic parameters for hydrolysis of benzyloxycarbonyl-phenylalanyl-arginyl-7(4-methyl)coumarylamide were determined for the three enzymes. Values for kcat/Km varied only within a factor of 2, but the individual constants were much higher for papain than for chymopapain and papaya proteinase III. In contrast to the results obtained with the synthetic substrate, the kinetic parameters for the initial hydrolysis of succinyl-albumin were very similar for the three papaya proteinases. This was consistent with their similar proteolytic activities in other assays.     

Stage-specific gut proteinases of the cotton stainer bug Dysdercus peruvianus: role in the release of entomotoxic peptides from Canavalia ensiformis urease

Canavalia ensiformis ureases are toxic to insects of different orders. The entomotoxicity of urease is due to a 10 kDa internal peptide released by proteinases in the insect digestive tract. We previously observed that, given orally, urease is toxic to nymphs of Dysdercus peruvianus, but does not affect adults. Here we characterized the major proteolytic activities of D. peruvianus midgut homogenates and investigated their in vitro-catalyzed release of the 10 kDa entomotoxic peptide from urease. Cysteine, aspartic and metalloproteinases are present in both homogenates. Variations in optimal pH and susceptibility to inhibitors indicated differences in the enzyme profiles in the two developmental stages. Only nymph homogenates released approximately 10 kDa fragment(s) from urease, recognized by antibodies against the entomotoxic peptide. Fluorogenic substrates containing urease partial sequences flanking the N-terminal or the C-terminal portion of the entomotoxic peptide were efficiently cleaved by homogenates from nymphs, but much more slowly by the adult homogenate. Different classes of enzymes in the homogenates cleaved both substrates suggesting that in vivo the release of the entomotoxic peptide results from the concerted action of at least two different proteinases. Our findings support the view that a differential processing of ingested urease by the insects explains at least in part the lack of toxicity in adults.