Recombinant histatins : functional domain repetition enhances candidacidal activity
Date
1995
DOI
Authors
Zuo, Yi
Version
OA Version
Citation
Abstract
Histatins are a family of small basic proteins found in human salivary secretions that are abundant in histidine residues. The fungicidal activity in particular has generated considerable interest.
Recent advances in molecular biology have made it possible to produce recombinant forms of the histatins and modified histatins to facilitate investigation of the fungicidal activity. However, prior to these studies a recombinant expression system for the production of biologically active histatins did not exist. Therefore, the major scientific problem addressed by this thesis was to establish a recombinant expression system for the production of biologically active histatins and histatin variants. To explore further structure/function relationship of histatins, we developed a bacterial system for the efficient production of recombinant histatin 3 (re-Hst3). Histatin 3 is a 32 amino acid member of the histatin family with antimicrobial activity found in human salivary secretions. Additionally, to gain insights into the mechanism of activity of histatins, we used the same system for the expression and purification of recombinant histatin variants. Previously, it was demonstrated that a middle portion of histatin 3 (residues 13-24) contains a functional domain responsible for killing of the pathogen yeast, Candida albicans, (C. albicans). Using polymerase chain reaction (PCR) and splice overlap extension (SOE), a recombinant variant(re-Hst3rep) was made in which the functional domain was repeated in tandem. Using the PRSET system, re-Hst3 and the variant re-Hst3rep were produced as chimeric fusions and were isolated from by affinity chromatography. Affinity purified fusion proteins were digested with cyanogen bromide and proteins were separated by reversed-phase high performance liquid chromatography. The activity of recombinant Hst3 and re-Hst3rep was compared to that of native histatin 3 in the C. albicans killing assay. The LD subscript 50 values for candidacidal activity of native histatin 3, re-Hst3 and re-Hst3rep were 7.2, 6.8, and 4.1 nmol/mL. At all doses re-Hst3rep demostrated candidacidal activity that was comparable to that of native histatin 3. At lower concentrations re-Hst3rep was five times more active than native Hst3 or re-Hst3 and at even lower concentrations re-Hst3ep exhibited significant candidacidal activity while native histatin 3 and re-Hst3 were inactive. These results demonstrate an expression system for production of biologically active re-Hst and re-Hst variants and shows that repetition of the functional domain enhances candacidal activity.
To investigate further the structural features of histatins with tandem repeats which confer candidacidal activity, an additional set of recombinant Hsts was produced using the same bacterial expression system. The set was designed to investigate the effect of re-Hsts bearing up to four tandemly repeated functional domains on candidacidal activity. The cDNA to express re-Hst having multiple functional domains were generated using a recent modification of the Taq DNA polymerase-mediated chain reaction, known as concatemer chain reaction (CCR), can produce tandemly repeated nucleotide sequences joined exactly gives greatly improved yields and is more efficient than conventional methods to produce concatemers. Cadidacidal assays indicated that these recombinant molecules were biologically active and exhibited dose-dependent killing. Further, activity was directly proportional to the number of functional domains contained within the recombinant molecule. The rack order was, such that reHst(4n) [more than]reHst(3n)[more than]reHst(2n)[more than]reHst3. Also, they extend the hypothesis that salivary proteins have adapted by evolution to contain amino acid repeats of selected regions which promote functional activity. Since the histatins appear very recently on the evolutionary tree, native molecules do not display these repeats.
The implications of these results on the structural features of histatins responsible for candidacidal activity, their evolutionary significance and impact on the therapeutic design of oral antimicrobials are discussed.
Description
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.
Thesis: (D.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1995 (Oral Biology).
Includes bibliographical references (leaves 100-114).
Thesis: (D.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1995 (Oral Biology).
Includes bibliographical references (leaves 100-114).
License
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.