Proteolysis of histatins in glandular secretions and whole saliva
Date
2004
DOI
Authors
Alagl, Adel S.
Version
OA Version
Citation
Abstract
Histatins are a family of small, cationic, histidine-rich proteins present in human salivary secretions (Oppenheim et al., 1988). The major ones are histatin 1 (38 amino acids), histatin 3 (32 amino acids) and histatin 5 (24 amino acids), which constitute 80% of total histatins in saliva. So far only two genes for histatins have been identified, the HIS 1 gene, encoding histatin 1 and the HIS 2 gene encoding histatin 3 (Sabatini and Azen, 1989). No gene for histatin 5 has been found despite the fact that its concentration in parotid secretion (PS) and submandibular/sublingual (SMSL) secretion is comparable to that of histatin 1 and 3.
In order to establish or predict histatin function in the oral cavity, it is important to know their concentrations in the various salivary secretions. Therefore, the first objective of this study is to isolate and quantify the major histatins in parotid secretion, submandibular/sublingual secretion, and in whole saliva. The recently developed zinc precipitation method was used (Flora et al., 2001) to obtain a fraction selectively enriched in histatins. The major histatins were subsequently separated by reversed-phase chromatographic procedures, and histatin concentrations were determined by comparison to standard curves for each of the histatins. The concentrations of the total major histatins in PS, SMSL and whole saliva supernatant (CHWS) from four individuals were found to range between 4.6 to 5.6 mg% in PS and from 9.8 to 18.4 mg% in SM/SL but only from 0.2 to 0.6 mg% in CHWS. Histatin levels were also compared in PS from a group of healthy individuals and periodontitis patients. The major histatin levels were higher in periodontitis patients than in healthy individuals but the differences were not statically significant due to large inter-individual variations within one group.
Notably, the concentrations of histatins in CHWS were much lower than the levels in pure glandular secretions. The second objective of this study was to investigate the reason for this. It was investigated whether histatins in whole saliva were bound to bacteria and cells rather than being present in a free form, the efficiency of the zinc precipitation method in CHWS was evaluated, and the susceptibility of histatins to proteolysis in CHWS was established. It can be concluded from our observations that histatins are removed from the solute phase of saliva by over 50% upon centrifugation, that with the zinc precipitation method, 86% of the histatins remaining in the solute phase can be precipitated and that histatins are quickly degraded by proteolytic enzymes in CHWS. All three factors likely contribute to the low histatin levels observed in CHWS.
The proteolytic degradation of histatins in CHWS was investigated in more detail by adding individual histatins to CHWS and by mixing PS or SMSL secretion with CHWS. To determine the primary cleavage sites in histatins, the histatin degradation patterns generated by CHWS were compared to degradation patterns generated by pure proteolytic enzymes that are known to be present in whole saliva. In particular histatin 3 and 5 added to CHWS were quickly degraded, and the major enzymes responsible for this appear to be trypsin and chymotrypsin. Interestingly histatin 1 was much more resistant to proteolysis than histatin 3 and 5.
Also in this study, the stability of native histatins in PS, SMSL and CHWS was investigated. In these experiments, PS, SMSL and CHWS samples were incubated individually for a 0-120 hr time interval at 37°C. At various time intervals, aliquots were removed, boiled and analyzed by cationic PAGE. Histatin
3 in both PS and SMSL started to disappear after an hour and was completely abolished after six hours of incubation. On the other hand, histatin 1 remained stable in both glandular secretions over the entire 120 hr incubation period. It can be concluded that PS and SM/SL secretion possess minor auto-proteolytic activities which are capable of degrading histatin 3 and 5 but not histatin 1.
The high resistance of native histatin 1 was an observation of interest. Since the amino acid sequences are very similar, the number of predicted proteolytic degradation sites in histatin 1 and 3 are virtually identical. It was investigated whether the proteolytic resistance of histatin 1 could be attributed to the phosphate group that is covalently linked to the amino acid serine at position 2 in histatin 1, but not in histatin 3. For this purpose, native, phosphorylated, histatin 1 and recombinant, non-phosphorylated, histatin 1 were incubated with CHWS and the proteolytic breakdown products were analyzed by cationic PAGE. Interestingly, while recombinant non-phosphorylated histatin 1 disappeared, native phosphorylated histatin 1 showed only minor losses. This result indicates that the presence of a phosphate group in position 2 in the 38 amino acid polypeptide structure of histatin 1 essentially protects this protein against proteolytic degradation.
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Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2004 (Oral Biology).
Includes bibliographical references (leaves 102-118).
Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2004 (Oral Biology).
Includes bibliographical references (leaves 102-118).
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This work is protected by copyright. Downloading is restricted to the BU community. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.