A Cascade of 24 Histatins (Histatin 3 Fragments) in Human Saliva

Abstract

The systematic search by tandem mass spectrometry of human saliva from four different subjects, of 136 possible fragments originated from histatin 3, allowed the detection of 24 different peptides. They include, with the exception of histatin 4, all the known histatin 3 fragments, namely histatins 5-12 and the peptides corresponding to 15-24, 26 -32, 29 -32 residues, and 13 new fragments corresponding to 1-11, 1-12, 1-13, 5-13, 6 -11, 6 -13, 7-11, 7-12, 7-13, 14 -24, 14 -25, 15-25, and 28 -32 residues of histatin 3. On the contrary, none of 119 possible fragments of histatin 1, including histatin 2, was detected. The results suggest that the genesis of histatin 3-related peptides, being under the principal action of trypsin-like activities, is probably not a random process but rather follows a sequential fragmentation pathway. Lack of detection of C-terminal fragments, with the exception of 26 -32, 28 -32, and 29 -32 fragments, suggested that arginine 25 should be the first cleavage site, generating histatin 6 and 26 -32 fragments. The genesis of 28 -32 and 29 -32 fragments and histatin 5 should implicate a subsequent exo-protease action. Similarly, lack of detection of fragments having Lys-5 and Arg-6 at the N terminus and Arg-25 at the C terminus strongly suggested that sequences KRKF (11-14 residues) and AKR (4 -6 residues) should be the second and the third cleavage sites, respectively. Lys-17 and Arg-22 are not cleaved at all.Histatins are a class of salivary peptides probably present only in higher primates (1), deriving their name from the high histidine content (2, 3). The powerful antifungal action of this class of peptides stimulated intense investigations concerning their properties, activity, structure, and secretion (4). Until now, only two human genes, HTN1 (HIS1) and HTN2 (HIS2), localized on chromosome 4q13, have been recognized as responsible for their synthesis (1, 5). The products of these two genes are histatin 1 and histatin 3, respectively. The former is a peptide of 38 amino acids, phosphorylated at Ser-2, whereas the latter, 32 amino acid long with a sequence very similar to histatin 1, is not phosphorylated. Many other peptides of this family have been identified in human saliva, all sharing a sequence common to the two parent peptides. Although different classifications have been proposed, the current preferred nomenclature derives from the study of Troxler et al. (6), who identified in human saliva a peptide corresponding to the C-terminal 26 residues of histatin 1, named histatin 2, and nine peptides, all related to the sequence of histatin 3 and named histatins 4 -12. Except for histatin 2, the other minor histatins likely originated by proteolytic cleavages from histatin 3. Among them, histatin 5, showing a sequence identical to the first 24 amino acids of histatin 3, represents the major fragment because it is present in human saliva at a higher concentration than the other fragments. Moreover, it appears to display the highest specific activity against Candida albicans species wit...

Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics

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

“…The gels were destained in the same solutions not containing the respective dyes until the band intensity was optimal relative to the background staining. These proteins have been demonstrated to be among the most sensitive to WS-associated proteolysis in prior studies (21,22,28,29). Fig.…”

Human body fluid proteome analysis

“…Both FFE and CIEF were successfully coupled with LIT-MS analysis, leading to the identification of 437 and 1381 proteins, respectively, in whole saliva [373,374]. In addition, a specific subgroup of saliva proteins such as proline-rich proteins (PRPs), histatins, and cystatins, were independently identified and characterized [375][376][377][378], and relative quantitation of saliva LMW peptides was also demonstrated using isotope tagging for relative and absolute quantitation (iTRAQ) and MALDI-TOF/TOF [379].…”