Histidine-rich Polypeptides Research Articles

Immunoreactive Histatin 5 in Salivary Gland Tumors.

Histatin is a group of histidine-rich polypeptides specific to parotid and submandibular gland secretions with several different biological functions such as stabilization of mineral-solute interaction in oral fluid, and antibacterial and antifungal actions. The authors generated polyclonal antibody to histatin by using purified histatin 5 as an immunogen and assayed the immunoreactivity by enzyme linked immunosorbent assay (ELISA) and immunoblotting. The antibody was further used to localize histatin in normal and tumors of salivary glans, pleomorphic adenoma, Warthin's tumor, adenoid cystic, acinic cell, mucoepidermoid, papillary cystadenocarcinoma and undifferentiated carcinoma by immunohistochemical methods. The normal major and minor human salivary glands showed an intense immunoreactivity in ductal cells, trace immunoreactivity in serous acini and no immunoreactivity in mucous acinar cells, suggesting histatin in mainly produced by ductal cells and to a lesser extent by serous cells. A consistent immunoreactivity of histatin in ductal segments of normal glands and a variable expression in the tumor cells of all the neoplastic lesions examined may implicate a role of this polypeptide in normal salivary gland function and salivary tumors. In addition, the findings may implicate common precursor cells, however, differing in the state of differentiation in normal and neoplastic conditions.

Anti-lipopolysaccharide activity of histatins, peptides from human saliva.

Histatins are histidine-rich polypeptides secreted in human saliva. They were found to inhibit lipopolysaccharide (LPS)-mediated gelation of Limulus amoebocyte lysate, and to reverse the anti-complement action of LPS or lipid A. Histatins also gave precipitate bands in agarose gels with various LPS. The results indicate that histatins neutralized the activity of LPS by binding to the lipid A moiety of LPS.

Salivary proteolysis of histidine-rich polypeptides and the antifungal activity of peptide degradation products

Incubation of purified synthetic histidine-rich polypeptides, HRP-2, -3, -4, -5, -6 (histatins), with diluted human parotid saliva yielded a series of peptide degradation products whose structures could be determined by gas-phase sequencing of cationic polyacrylamide gel electroblots. Sequencing indicated that two and sometimes three peptides were present in the same Coomassie blue-stained band. By comparing different individuals' salivas it was observed that structural variation occurs, perhaps due to differences in the concentrations or specific activities of salivary proteases. Based on the structural data, four proteolytic enzyme activities are proposed. A trypsin-like and chymotrypsin-like enzymatic activity(s) appear to represent the most active salivary protease; however, both an alanine-lysine endopeptidase and a histidine peptidase activity are also present in parotid saliva. In comparison to HRP-4 or HRP-6, degraded products were less active as antifungal agents against Candida albicans both in blastospore and germ-tube assays.

Histatins 2 and 4 are autoproteolytic degradation products of human parotid saliva.

Freshly collected human parotid saliva contains 8 cationic proteins, as demonstrated by capillary electrophoresis. These proteins include lysozyme, histatin 6 and the 6 salivary histidine-rich polypeptides (HRPs 1-6). Neither histatin 2 nor histatin 4 are present in native undegraded parotid saliva but appear only after autoproteolytic degradation of the saliva. Histatin 2 appears to arise through slow degradation of HRP-1, and histatin 4 is mainly produced as a rapid breakdown product of HRP-3.

One-step purification of histidine-rich polypeptides form human parotid saliva and determination of anti-candidal activity.

Immunoadsorption affinity chromatography was used to selectively purify the family of the histidine-rich polypeptides (HRPs) from human parotid saliva. The immunoadsorbent was prepared by coupling an enriched preparation of horse anti-(HRPs 2, 3, 4, 5 and 6) IgG to protein G. Both freshly collected stimulated untreated and acidified boiled salivas (5 ml) were applied to the affinity column. When native saliva was used it appeared that all of the components of saliva, with the exception of the HRPs, were present in the fraction nonadsorbed to the affinity column; however, recovery of the HRPs with 0.2 M sodium acetate-HCl, pH 1.8, was poor. Yields of HRPs desorbed from the column with the pH 1.8 treatment were significantly improved if salivary HRP proteolysis was delayed immediately after collection by acidifying the saliva to pH 4.5 followed by a short boiling time period, which neither affected HRP quantification nor biological activity. Affinity chromatography results were checked both by cationic polyacrylamide gel and by capillary electrophoresis. Antifungal activity was found to reside only in the low pH HRP fraction of the immunoadsorbent column, suggesting that it is the histidine-rich family of polypeptides that is responsible for salivary antifungal action.

In vivo antifungal efficacy of salivary histidine-rich polypeptides: Preliminary findings in a denture stomatitis model system

Six denture stomatitis patients, all found to have Candida albicans on their maxillary denture and palatal tissue surfaces, volunteered in this preliminary study to test the in vivo efficacy of human salivary antifungal histidine-rich polypeptides (HRPs) in treating their oral disease. The patients were equally divided among the Newton types classification and, as expected, the severity of the inflammation was greatest in the Newton type III patients and least in the Newton type I patients. Patients received sterile solutions of either HRP-3 or HRP-4, which they used both as a mouthrinse and as a denture soak for a period of 1 week. Agar replicas of the tissue-fitting surface of the maxillary dentures revealed HRP reduction and/or elimination of C. albicans from the denture; in one Newton type II individual, this finding directly correlated with a site-specific reduction in palatal inflammation. In the Newton type II and type III individuals alike, there was a significant generalized decrease in inflammation suggesting the therapeutic efficacy of the HRPs. Killing of this yeast species by the HRPs, as determined by scanning electron microscopy (SEM), was probably responsible for the observed clinical benefits noted in this investigation. In the SEM, HRP-treated blastospores appeared severely deflated, as if they had been emptied of significant quantities of intracellular material.

Selective effects of histidine‐rich polypeptides on the aggregation and viability of Streptococcus mutans and Streptococcus sanguis

Enriched preparations of histidine-rich polypeptides (HRPs) and isolated HRP pairs (1-2, 3-4 and 5-6) degrade in the presence of fresh autologous whole saliva to a series of low-molecular-weight cationic peptides (HRPs 6a-c and 7). Analysis of the HRPs during degradation indicates that: HRP 1 is not the parent molecule of the HRPs; the HRP pairs do not convert to each other in a cascade-like sequence in saliva; and the HRPs can be separated into 2 groups consisting of HRPs 1-2 and 3-7. Preparations containing HRPs 1-7, 1-2, and 3-7 were obtained by fractionation and separation on Bio-Rex 70, and tested for aggregating and antibacterial effects against Streptococcus mutans BHT, S. mutans GS-5 and Streptococcus sanguis G9B. HRPs 1-2 had significant aggregating effects on all 3 strains but the other HRPs had little to no agglutinating ability. The HRPs did not inhibit the growth of S. sanguis, and HRPs 1-2 enhanced its growth. No growth enhancement by the HRPs was observed for the 2 S. mutans strains. However, significant bacterial inhibition of the S. mutans strains was noted after incubation with HRPs 3-7. The data suggest that the dissimilar effects of HRPs 1-2 and 3-7 may be of importance in the colonization and growth of S. mutans and S. sanguis in vivo.

The role of pH in salivary histidine‐rich polypeptide antifungal germ tube inhibitory activity

Purified synthetic salivary histidine-rich polypeptides (HRPs) 2, 3, 4, 5 and 6 were found to inhibit Candida albicans conversion of blastospores to germ tubes. HRP-4 was the best inhibitor within the pH 5 to 7 range tested and all of the HRPs were observed to lose potency as the pH was raised from 5 to 7. The pH pattern obtained with a synthetic homologous histidine peptide suggested that the protonated form of the histidine imidazole residues of the HRPs was important to the germ tube antifungal activity. Similar pH inhibition profiles of germ tube formation by parotid saliva and the HRPs were also observed.

Parameters affecting the inhibition of Candida albicans GDH 2023 and GRI 2773 blastospore viability by purified synthetic salivary histidine‐rich polypeptides

Purified synthetic salivary histidine-rich polypeptides, HRPs 2, 3, 4, 5, and 6, were observed to inhibit Candida albicans blastospore viability at yeast cell concentrations ranging from 10(2) to greater than 10(6) colony forming units per ml. Among the HRPs, HRP-4 was the best inhibitor with significant killing activity noted at a peptide concentration of 0.5 microgram per ml. Antifungal potency under growth conditions was observed to be dependent upon pH. In contrast, killing did not vary throughout the pH range tested under non-growth conditions. Electron microscopy results demonstrated HRP damage at pH 5 which appeared to be initiated at the membrane. At pH 7.4, micrographs revealed clear evidence of intracellular destruction suggesting more extensive damage at neutral as compared to acidic pH. These results suggest that within the changing realm of the oral cavity, the HRPs would be expected to be potent killers of C. albicans.

Rapid purification and characterization of histatins (histidine-rich polypeptides) from human whole saliva

Three different polypeptides capable of stimulating histamine release from mast cells were isolated from human whole saliva, using heparin-gel chromatography followed by reversed-phase HPLC. The amino acid sequences of these peptides were shown to be identical to those of histatins 1, 3 and 5.

Structural Relationship Between Human Salivary Histatins

Histatins are a group of electrophoretically distinct histidine-rich polypeptides with microbicidal activity found in human parotid and submandibular gland secretions. Recently, we have shown that histatins 1, 3, and 5 are homologous proteins that consist of 38, 32, and 24 amino acid residues, respectively, and that these polypeptides kill the pathogenic yeast, Candida albicans. We now describe the isolation and structural characterization of histatins 2, 4, 6, and 7-12, the remaining members of this group of polypeptides. Histatin 2 was found to be identical to the carboxyl terminal 26 residues of histatin 1; histatin 4 was found to be identical to the carboxyl terminal 20 residues of histatin 3; and histatin 6 was found to be identical to histatin 5, but contained an additional carboxyl terminal arginine residue. The amino acid sequences of histatins 7-12 formally correspond to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively, of histatin 3, but could also arise proteolytically from histatin 5 or 6. These results establish, for the first time, the complete structural relationships between all members of this group of microbicidal proteins in human parotid saliva. The relationship of histatins to one another is discussed in the context of their genetic origin, biosynthesis and secretion into the oral cavity, and potential as reagents in anti-candidal studies.

Molecular cloning of human submandibular histatins

Histatins are a group of histidine-rich polypeptides found in human parotid and submandibular gland secretions. These polypeptides are microbiocidal, possibly involved in maintaining the acquired enamel pellicle, and enhance the glycolytic activity of certain oral micro-organisms. Histatins 1, 3 and 5 are homologous proteins with 38, 32 and 24 amino acid residues, respectively; the cDNAs coding for histatins 1 and 3 have now been isolated and sequenced. The cDNA sequences were highly homologous but contained differences throughout their length, indicating that they arise from different genes that may be derived from a common ancestral gene. Northern blots were hybridized to a series of oligonucleotide probes, designed on the basis of histatin cDNA sequences, and these positively identified mRNAs for histatins 1 and 3. In addition, there was a third mRNA, which hybridized to several histatin oligonucleotide probes, suggesting that histatin 5 might be derived from a distinct mRNA and not by proteolytic processing of histatin 3. A Northern blot of macaque parotid gland total RNA also showed three histatin mRNAs, indicating that similar histatins exist in a non-human primate.

Pellicle receptors for Actinomyces viscosus type 1 fimbriae in vitro

Actinomyces viscosus T14V-J1 and its fimbria-deficient mutant strain possessing type 1 fimbriae strongly aggregated with latex beads treated with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative. These type 1+ strains did not aggregate with latex beads treated with other proteins, such as salivary amylase, salivary histidine-rich polypeptides, laminin, type 1 collagen, fibronectin, or C1q. The type 1+ strains also adsorbed well to experimental pellicles formed with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative on hydroxyapatite (HA) surfaces. These interactions were inhibited with immunoglobulins and Fabs specific for type 1 fimbriae. Type 1- actinomyces exhibited feeble adsorption to latex beads or HA treated with any of the aforementioned proteins. Collectively, these data indicate that actinomyces type 1 fimbriae may specifically interact with several proline-rich salivary molecules, forming experimental pellicles on HA or polystyrene surfaces.

P-050 - Histamine release induced by histatins (histidine-rich polypeptides in human saliva) from rat mast cells

P-050 - Histamine release induced by histatins (histidine-rich polypeptides in human saliva) from rat mast cells

Histidine-rich domain of the knob protein of the human malaria parasite Plasmodium falciparum.

Membranes of erythrocytes infected with the human malaria parasite Plasmodium falciparum develop protrusions called knobs. These structures are essential for the survival of the parasite in the host, and their induction requires the synthesis of the knob protein by the parasite. We describe the isolation of a cDNA clone encoding the amino-terminal half of the knob protein. A cDNA library was constructed from RNA prepared from ring stages of a P. falciparum isolate that has retained its ability to induce knobs (knob+ phenotype). A synthetic oligonucleotide probe encoding polyhistidine was used to isolate the cDNA clone, which encodes the amino-terminal half of a polypeptide with all the known attributes of the knob protein. The gene is not transcribed in variants that do not synthesize the knob protein and thereby cannot induce knobs (knob- phenotype). The apparent lack of transcription in knob- variants is due to different mechanisms: although the gene is present in one knob- isolate, it has been deleted in a cloned knob- variant. The primary structure of the polypeptide deduced from a partial sequence of the cDNA is distinctly different from other malarial histidine-rich polypeptides. The amino-terminal sequence shows the characteristic features of a signal peptide. This is followed by a histidine-rich domain and a subsequent region which contains one histidine. Peptide map analysis of the knob protein is consistent with the structural features deduced from the sequence analysis of the cDNA.

The amino acid sequence of a salivary peptide (F-A) with histamine-releasing property.

We have found that human parotid saliva contained the substances responsible for releasing histamine. They were basic peptides whose histidine contents were extremely high. Human parotid saliva has previously been shown to contain a group of histidinerich polypeptides (HRPs). However, their function is unclear and the sequence of the individual HRPs have not been determined. In this report, we further purified one of the HRPs, F-A with histamine-releasing property and estimated its amino acid sequence.

Fungistatic and fungicidal activity of human parotid salivary histidine-rich polypeptides on Candida albicans.

Human parotid saliva histidine-rich polypeptides exerted antifungal activity against Candida albicans at concentrations similar to the known antifungal activity of the imidazole antibiotics. Inhibition of both growth and viability could be demonstrated by optical density monitoring and plating assays. Inhibition of growth was observed to be greatest when the histidine-rich polypeptides were added to the inoculum before addition to the growth media. However, complete inhibition by these polypeptides was still noted during active growth at turbidities of C. albicans corresponding to 10(6) CFU/ml. At higher cell densities, growth was delayed but not halted under the experimental conditions investigated. Candidacidal activity was observed with both growing and nongrowing cells. With respect to the latter, reaction of cells in buffer with the histidine-rich polypeptides for a period of 30 min resulted in killing of greater than 90% of two different strains of C. albicans, whereas a third strain was found to be less susceptible. Moreover, the kinetics of loss of cell viability correlated with the loss of potassium from the cells. In addition to the histidine-rich polypeptides, hen egg white lysozyme, poly-L-lysine, and poly-L-histidine affected C. albicans. Both of the polyamino acids completely inhibited the growth of the yeast whereas lysozyme was not as potent. Where delays in growth were observed for all of these agents, including the histidine-rich polypeptides, turbidities reached those of untreated controls after a 24-h period. Enhanced effects were noted if C. albicans was preincubated with these agents in 0.025 2-(N-morpholino)-ethanesulfonic acid buffer, pH 5.2, before growth in the yeast synthetic medium.

Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans.

Growth inhibition and cell viability assays demonstrate that the histidine-rich polypeptides isolated from human parotid saliva are bacteriostatic and bactericidal for strains of Streptococcus mutans belonging to the serotype b and c classifications. Both inhibition of growth and cell division are enhanced by preincubation of bacteria with these polypeptides in low-ionic-strength buffers of acidic and neutral pH before dilution into enriched growth media. With prior exposure at pH 6.8, inhibition by these polypeptides of the serotype c strains, S. mutans GS5 and SB, as well as the serotype b strain, S. mutans BHT, is reversible over time under the experimental conditions selected. With similar exposure at pH 5.2, however, irreversible damage is manifested by complete inhibition of both growth and cell viability. At concentrations of 250 micrograms of the mixture of histidine-rich polypeptides per 5 X 10(5) bacterial cells per ml in the acidic preincubation buffer, bacterial lethality is maintained for a period of 48 h in the enriched growth media. At a 50-micrograms/ml concentration of these salivary agents, approximately 80% killing of S. mutans SB is noted after a 24-h incubation; however, surviving bacteria multiply and reach turbidities of untreated control cells when examined at the 48-h growth point. Similarly, hen egg white lysozyme is also found to be bactericidal for these microorganisms when preincubation is carried out under acidic conditions. However, in contrast to the histidine-rich polypeptides, lysozyme under these experimental conditions does not inhibit growth of S. mutans SB at neutral pH, although it does inhibit growth of both S. mutans BHT and S. mutans GS5 at this pH. Preexposure of S. mutans SB to the peptides in buffer at ionic strengths of 0.025 to 0.125, followed by either viability assays under nongrowing conditions or growth inhibition studies, suggests that there is very little effect of ionic strength on the antibacterial function of these peptides. In contrast to the inhibition of viability noted under growing conditions, lower concentrations of the histidine-rich polypeptides were required to elicit immediate cell death under nongrowing conditions.

Isolation of milligram quantities of a group of histidine-rich polypeptides from human parotid saliva.

Freshly collected parotid saliva collected from human donors were shown by polyacrylamide gel electrophoresis to continuously secrete a group of low-molecular-weight cationic polypeptides. Up to 14 bands could be identified by Coomassie blue staining, and all bands migrated more rapidly than purified human leukemic lysozyme in cationic polyacrylamide gel electrophoresis. These peptides could be isolated as a group relatively free of other salivary components and recovered in high yields from concentrated parotid saliva by Sephadex G-25 chromatography. In sodium dodecyl sulfate gel electrophoresis, the histidine-rich polypeptide bands appeared as just two bands migrating at the tracking dye and ahead of insulin chain B. Amino acid analysis of the mixture revealed an average content of at least 48% cationic residues, of which half were histidine. When stained bands were eluted from electrophoretic gels, hydrolyzed, and subjected to amino acid analyses, they were found to be enriched in histidine. There was also a correlation of the electrophoretic mobility with the content of basic amino acids. Sephadex G-25 chromatography is a convenient, simple method for preparing milligram quantities of the histidine-rich polypeptides for chemical and biochemical studies.

Evidence that the Histidine-Rich Polypeptides of Human Parotid Saliva are Acinar Secretory Products of the Parotid Gland

Evidence that the Histidine-Rich Polypeptides of Human Parotid Saliva are Acinar Secretory Products of the Parotid Gland