A New Method for the Isolation of Histatins 1, 3, and 5 from Parotid Secretion Using Zinc Precipitation

Flora, Bianca; Gusman, Heloísa; Helmerhorst, Eva J.; Troxler, Robert F.; Oppenheim, Frank G.

doi:10.1006/prep.2001.1493

Cited by 40 publications

(39 citation statements)

References 34 publications

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“…The absorbance in this region is substantially lower in WS than in PS (see Fig. 4) or in SMSL secretion (30). Masticatory-stimulated WS was more similar to PS than unstimulated WS, consistent with the fact that masticatory stimulation increases PS output and reduces the residence time of proteins in the proteolytic environment of the oral cavity.…”

Section: Methodssupporting

confidence: 63%

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Identification of Lys-Pro-Gln as a Novel Cleavage Site Specificity of Saliva-associated Proteases

Helmerhorst

Sun

Salih

et al. 2008

Journal of Biological Chemistry

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The nonsterile environment of the oral cavity facilitates substantial proteolytic processing, not only of resident salivary proteins but also of dietary proteins. To gain insight into whole saliva enzymatic processes, the in vivo generated peptides in this oral fluid were subjected to nano-flow liquid chromatography electrospray ionization tandem mass spectrometry. The 182 peptides identified were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins. The proteolytic cleavages in the basic proline-rich proteins occurred preferentially after a Gln residue with predominant specificity for the tripeptide Xaa-Pro-Gln, where Xaa in the P 3 position was mostly represented by Lys. Using the synthetic substrates LysPro-Gln-pNA and Gly-Gly-Gln-pNA, the overall K m values were determined to be 97 ؎ 7.7 and 611 ؎ 28 M, respectively, confirming glutamine endoprotease activity in whole saliva and the influence of the amino acids in positions P 2 and P 3 on protease recognition. The pH optimum of Lys-Pro-Gln-pNA hydrolysis was 7.0, and the activity was most effectively inhibited by antipain and 4-(2-aminoethyl) benzenesulfonyl fluoride, was metal ion-dependent, and not inhibited by cysteine protease inhibitors. A systematic evaluation of enzyme activities in various exocrine and nonexocrine contributors to whole saliva revealed that the glutamine endoprotease is derived from dental plaque and likely microbial in origin. The P 1 site being occupied by a Gln residue is a nonarchetype with respect to known proteases and indicates the presence of novel glutamine-specific endoprotease(s) in oral fluid.

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Section: Methodssupporting

confidence: 63%

“…1A shows the C 18 RP-HPLC chromatographic comparison of proteins present in equal volumes of PS, stimulated WS supernatant, and unstimulated WS supernatant. Intact acidic PRPs, histatins, and statherin elute between 35 and 55 min using the gradient applied (30). The absorbance in this region is substantially lower in WS than in PS (see Fig.…”

Section: Methodsmentioning

confidence: 97%

Identification of Lys-Pro-Gln as a Novel Cleavage Site Specificity of Saliva-associated Proteases

Helmerhorst

Sun

Salih

et al. 2008

Journal of Biological Chemistry

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“…Salivary protein substrates used were synthetic histatin 5 (American Peptide Company, Sunnyvale, CA, USA), statherin, or PRP1, which were both isolated from parotid secretion as described previously (Oppenheim et al, 1982;Flora et al, 2001). All proteins were > 90% pure as determined by gel electrophoresis and chromatography (data not shown).…”

Section: Effect Of An Inhibitor Cocktail On Protein Degradation In Wsmentioning

confidence: 99%

“…Cationic PAGE and densitometric analysis of the histatin 5 band were performed as described previously (Baum et al, 1977;Flora et al, 2001). The intensity of the histatin 5 band at t = 0 (immediately after addition to WS) was set to 100%.…”

Section: Cationic Polyacrylamide Gel Electrophoresis (Cationic Page) mentioning

confidence: 99%

Whole-saliva Proteolysis and Its Impact on Salivary Diagnostics

et al. 2011

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There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins—histatin 5, statherin, and PRP1—was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.

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“…Further purification efforts using gel filtration followed by reverse-phase high-performance liquid chromatography finally isolated three major distinct purified components consisting of Hsts 1, 3, and 5 (1). Subsequently, a novel and less laborintensive purification method that exploited the metal binding ability of Hsts, using zinc precipitation under alkaline conditions, was developed (7).…”

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confidence: 99%

How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5

2014

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Histatins are salivary cationic peptides that provide the first line of defense against oral candidiasis caused by Candida albicans. This minireview presents a critical evaluation of our knowledge of the candidacidal mechanism of histatin 5 (Hst 5). Hst 5 is the most potent among all histatin family members with regard to its antifungal activity. The mode of action of Hst 5 has been a subject of intense debate. Unlike other classical host innate immune proteins, pore formation or membrane lysis by Hst 5 has largely been disproven, and it is now known that all targets of Hst 5 are intracellular. Hst 5 binds C. albicans cell wall proteins (Ssa1/2) and glycans and is taken up by the cells through fungal polyamine transporters in an energy-dependent manner. Once inside the fungal cells, Hst 5 may affect mitochondrial functions and cause oxidative stress; however, the ultimate cause of cell death is by volume dysregulation and ion imbalance triggered by osmotic stress. Besides these diverse targets, a novel mechanism based on the metal binding abilities of Hst 5 is discussed. Finally, translational approaches for Hst 5, based on peptide design and synergy with other known drugs, are considered a step forward for bench-to-bed application of Hst 5.

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A New Method for the Isolation of Histatins 1, 3, and 5 from Parotid Secretion Using Zinc Precipitation

Cited by 40 publications

References 34 publications

Identification of Lys-Pro-Gln as a Novel Cleavage Site Specificity of Saliva-associated Proteases

Identification of Lys-Pro-Gln as a Novel Cleavage Site Specificity of Saliva-associated Proteases

Whole-saliva Proteolysis and Its Impact on Salivary Diagnostics

How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5

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