MS characterization of multiple forms of alpha-amylase in human saliva

Alpha-amylase is a major and well-characterized component of human saliva. Recent proteomic studies suggested that this protein could be observed in more than twenty spots on 2-D gels of salivary proteins. The aim of this work was to investigate this unexpected redundancy. 2-D gel electrophoresis was combined with systematic MALDI-TOF MS analysis. More than 140 protein spots identifying the alpha-amylase were shown to constitute a stable but very complex pattern. Careful analysis of mass spectra and simultaneous hierarchical clustering of the observed peptides and of the electrophoretic features of spots allowed one to define three major groups. A main class grouping 90 spots was shown to correspond to full length alpha-amylases that can be assumed to include isoforms and post-translationally modified forms, a subset of this class being demonstrated to be N-glycosylated. A second group included short alpha-amylases that are differently truncated in a non-random manner, very likely in the oral cavity. The last class grouped alpha-amylase forms showing both the N- and C-terminal sequences of the enzyme but displaying a molecular weight that was up to 50% lower than that of the native protein. It is speculated that the last group of alpha-amylase spots could correspond to proteins submitted to internal deletions prior to the secretion.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).     

The role of glandular kallikrein in the formation of a salivary proline-rich protein A by cleavage of a single bond in salivary protein C.

An enzyme was purified from human parotid saliva that can cleave a single arginine-glycine peptide bond between residues 106 and 107 in human salivary proline-rich protein C, hereby giving rise to another proline-rich protein A, which is also found in saliva. The enzyme was purified 2400-fold. It cleaved salivary protein C at the rate of 59 micrograms of protein/h per microgram of enzyme and had amino acid composition, molecular weight and inhibition characteristics similar to those reported for human salivary kallikrein. Confirmation that the enzyme was kallikrein was demonstrated by its kinin-generating ability. Histochemical evidence indicates that a post-synthetic cleavage of protein C by kallikrein would have to take place during passage of saliva through the secretory ducts. In secreted saliva, cleavage of salivary protein C can only be observed after 72 h incubation. In addition, there is no effect of salivary flow rate on the relative amounts of proteins A and C in saliva. On the basis of the experimental observations, it is proposed that in vivo it is unlikely that kallikrein secreted from ductal cells plays a significant role in converting protein C into protein A.     

Changes in proteolytic activities of human leukemic promyelocytes (HL-60 cells) during maturation

Proteolytic activity was measured in human leukemic promyelocytic cell line (HL-60) grown in culture, before and after the addition of agents which promote differentiation. The 36000 X g soluble fraction of the cells degraded [14C]globin with maximal activity at pH 3.6, while the insoluble fraction had a pH optimum at 8.0. This pattern did not change upon differentiation. The acid protease activity of the soluble fraction increased following differentiation. After 4 days in the presence of dimethyl sulfoxide, the differentiated cells exhibited 4-fold higher specific activity as compared with 4 day-old control cells. In contrast, the alkaline activity of the insoluble fraction of the differentiated cells was 4-fold lower than that of the undifferentiated cells. It is suggested that the changes in enzyme activities may serve the new functions acquired by the mature granulocytes.     

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD⁺ dependent enzyme which was inactivated by trypsing and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.     

Expression of human salivary alpha-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence

A cDNA fragment coding for human salivary alpha-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 X 10(5) molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as alpha-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.     

Insoluble complex formation between alpha-amylase from Aspergillus oryzae and polyacrylic acid of different molecular weight

The insoluble complex formation between alpha-amylase and the strong anionic polyelectrolyte polyacrylic acid was studied by using turbidimetric and enzymatic activity. The highest molecular weight polyacrylic acid (100,000 Da and 240,000 Da) proved to be suitable precipitating agents. They were insoluble at pH lower than 4–5, with a stoichiometric ratio polymer mol per protein mol of 1:52 and 1:154, respectively. Electrostatic interactions are not the only factor in the formation of insoluble complexes. High percentage of alpha-amylase enzymatic activity maintains throughout time, even in the presence of polyelectrolyte.The application of precipitation conditions found when applying a bovine homogenate showed that it is not suitable for purification even if it proved to be useful methodology for the concentration of the enzyme and can be used as a first step of purification.     

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.     

Triatoma infestans apyrases belong to the 5'-nucleotidase family

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5'-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5'-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5'-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.     

Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

Endopeptidase in human cerebrospinal fluid which cleaves proenkephalin B opioid peptides at consecutive basic amino acids

An endopeptidase releasing the common N-terminal hexapeptide, (Leu)-enkephalin-Arg6, from dynorphins A and B, and alpha-neoendorphin was purified from human cerebrospinal fluid. Purification involved ion-exchange chromatography (DEAE-Sepharose CL-6B), hydrophobic interaction chromatography (phenyl-Sepharose CL-4B) and molecular sieving (Sephadex G-100). The enzyme showed molecular heterogeneity. A major fraction had an apparent molecular weight of about 40,000. It had an optimum activity in the pH range of 6-8. The conversion of dynorphin A was not affected by EDTA or iodoacetate but strongly reduced in the presence of phenylmethyl-sulphonyl fluoride, suggesting the enzyme is a serine protease.     

Salivary film expresses a complex, macromolecular binding site for Streptococcus sanguis

Teeth in the oral cavity are coated with a salivary film or pellicle, which lacks apparent intermolecular organization. This heterogeneous film facilitates binding of early commensal colonizing bacteria, including Streptococcus sanguis. To test the hypothesis that sufficient intermolecular organization exists in salivary films to form binding sites for S. sanguis, an in vitro model of saliva-coated teeth was probed with murine anti-idiotypical monoclonal antibodies (mAb2, anti-ids). The anti-ids were harvested from hybridomas that were developed in response to first generation murine hybridomas that produced anti-S. sanguis adhesin monoclonal antibodies (mAb1). The anti-ids (i) reacted with experimental salivary films and inhibited S. sanguis adhesion in a dose-dependent fashion. In Western blots, the anti-ids (ii) recognized a high molecular weight salivary antigen and (iii) secretory IgA (sIgA) light chain and alpha-amylase. After isolation by gel filtration from whole saliva or mixed secretory IgA and alpha-amylase, the high molecular weight component, containing amylase activity and sIgA, bound to hydroxyapatite to promote adhesion of S. sanguis. Therefore, a complex enriched in secretory immunoglobulin A and alpha-amylase forms a S. sanguis-binding site.     

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.     

Measurement of pancreatic and salivary alpha-amylase in serum: comparison of methods with five different substrates

Pancreatic and salivary alpha-amylase in human serum have been determined with the inhibitor method with 5 different substrates. An end concentration of 60 inhibitor units per liter reagent is sufficient for inhibition of the salivary alpha-amylase. The recovery of known amounts of pancreatic and salivary alpha-amylase was excellent. A mean +/- SD of 53.1 +/- 3.8% of pancreatic alpha-amylase as percentage of total alpha-amylase in serum has been observed for the 5 different substrates used. Moreover, it seems that standards obtained from crude saliva or duodenal fluid can be used as well as pure enzymes obtained from a commercial supplier.     

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Identification and characterization of tyrosylprotein sulfotransferase from human saliva

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl2 for its activity. Maximum TPST activity was observed at 20 mM MnCl2. The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.     

Interaction of human alpha-amylases with inhibitors from wheat flour

The interaction of four purified alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) inhibitors with human salivary and pancreatic alpha-amylases was investigated. The inhibitory activity of the four proteins towards salivary alpha-amylase was significantly increased by pre-incubation of the enzyme with inhibitor before adding substrate. This effect was not observed with the inhibition of pancreatic alpha-amylase by inhibitors 1 and 2. Inhibition of both amylases was affected to different degrees by incubating starch with inhibitor prior to the addition of enzyme. Maltose, at concentrations which only slightly affected amylase activity, prevented the inhibition of both enzymes by all four inhibitors. Gel filtration studies on salivary amylase-inhibitor mixtures showed the formation of EI complexes on a mol-to-mol ratio. A similar complex between pancreatic alpha-amylase and inhibitor 4 was observed, though complex formation between pancreatic alpha-amylase and the other inhibitors was not clearly demonstrated.