Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability of Candida albicans cells was studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion.
Wounds in the oral cavity heal much faster than skin lesions. Among other factors, saliva is generally assumed to be of relevance to this feature. Rodent saliva contains large amounts of growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF). In humans, however, the identity of the involved compounds has remained elusive, especially since EGF and NGF concentrations are approximately 100,000 times lower than those in rodent saliva. Using an in vitro model for wound closure, we examined the properties of human saliva and the fractions that were obtained from saliva by high-performance liquid chromotography (HPLC) separation. We identified histatin 1 (Hst1) and histatin 2 (Hst2) as major wound-closing factors in human saliva. In contrast, the d-enantiomer of Hst2 did not induce wound closure, indicating stereospecific activation. Furthermore, histatins were actively internalized by epithelial cells and specifically used the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, thereby enhancing epithelial migration. This study demonstrates that members of the histatin family, which up to now were implicated in the antifungal weaponry of saliva, exert a novel function that likely is relevant for oral wound healing.
Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/ DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300 -400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.
The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteriabinding protein loop (peptide SRCRP2; QGRVEVLYRG-SWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.The scavenger receptor cysteine-rich (SRCR) 1 proteins form an archaic group of metazoan proteins (1-5). This group of glycoproteins comprises cell surface molecules as well as secreted proteins that are characterized by the presence of one or more SRCR domains. SRCR domains consist of ϳ110 amino acids and are divided into groups A and B based on the number of conserved cysteine residues, namely six for group A and eight for group B.The best studied members of the group A SRCR proteins are the macrophage scavenger receptor (MSR1), the Mac 2-binding protein (Mac-2bp), and MARCO. Both MSR1 and MARCO are known to interact with bacteria (6, 7). In contrast to MARCO (8), the SRCR domain of MSR1 does not seem to be involved in bacteria binding (9, 10). Bacteria binding by MARCO involves an RXR motif within the SRCR domain, indicating that ionic interactions play a crucial role in the interaction with its negatively charged ligands (6).Group B SRCR proteins are generally involved in the regulation of cellular immune responses. In vertebrates, the group B SRCR proteins can be divided, on the basis of their structure and sequence homology, into three subgroups (11). The first subgroup includes CD5 (12), CD6 (13), and SP␣ (14). CD5 and CD6 are composed of an extracellular region of three SRCR domains, a transmembrane region, and a cytoplasmic region. SP␣ lacks the latter two regions but contains three SRCR domains that are highly homologous to those of CD5 and CD6. These three proteins are mainly expressed by T-cells and Bcells (12, 13). The second subgroup of SRCR group B molecules is the workshop cluster 1 (WC1) family, which includes WC1, ...
Wounds in the mouth heal faster and with less scarification and inflammation than those in the skin. Saliva is thought to be essential for the superior oral wound healing, but the involved mechanism is still unclear. We have previously discovered that a human-specific peptide, histatin, might be implicated in the wound-healing properties of saliva. Here we report that histatin enhances reepithelialization in a human full-skin wound model closely resembling normal skin. The peptide does not stimulate proliferation but induces cell spreading and migration, two key initiating steps in reepithelialization. Activation of cells by histatin requires a G-protein-coupled receptor that activates the ERK1/2 pathway. Using a stepwise-truncation method, we determined the minimal domain (SHREFPFYGDYGS) of the 38-mer-parent peptide that is required for activity. Strikingly, N- to C-terminal cyclization of histatin-1 potentiates the molar activity approximately 1000-fold, indicating that the recognition of histatin by its cognate receptor requires a specific spatial conformation of the peptide. Our results emphasize the importance of histatin in human saliva for tissue protection and recovery and establish the experimental basis for the development of synthetic histatins as novel skin wound-healing agents.
The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC 50 /IC 50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC 50 = 0.6 W WM) and had the lowest therapeutic index (HC 50 / IC 50 = 0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.z 1999 Federation of European Biochemical Societies.
Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an ␣-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.
Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed.
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