Structural & functional characterization of the amyloid -like glycoprotein adhesin Als5p of Candida albicans.
Item
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Title
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Structural & functional characterization of the amyloid -like glycoprotein adhesin Als5p of Candida albicans.
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Identifier
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AAI3288883
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identifier
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3288883
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Creator
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Otoo, Henry Nunoo.
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Contributor
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Adviser: Peter N. Lipke
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Date
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2007
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Microbiology
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Abstract
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Candida albicans is the third most common nosocomial agent in the United States. The fungus expresses a family of cell wall proteins called Als (Agglutinin-Like Sequence proteins). Als proteins share a similar immunoglobulin-like (Ig), conserved Threonine-rich (T), variable Tandem repeats (TR), and glycosylated C-terminal regions. Als5p mediates adhesion to host and self aggregation.;With increasing TR number, binding of Als5p to ligands is enhanced (Rauceo et al., 2006). Biochemical studies done investigated the structural basis for this region's influence on aggregation (TR). Als5p1-431 (Ig-T) and Als5p1-664 (Ig-T-TR) were successfully purified in good quantities (4-10ml of 0.1-0.4mg/ml). Circular Dichroism indicates Als5p1-664 fragment has high beta sheet content and that the TR-region influences the conformation of Als5p1-664. The TR is shown to give Als5p1-664 conformational flexibility.;Als5p mediates aggregation at pH 2-10 and temperatures 5°C-65°C. Soluble Als5p aggregates at low concentrations (<0.5mg/ml) into multimers that are resistant to GuHCl and SDS dissolution. The soluble protein fragment is stable within the pH range 3-9, refolds from 80°C, retains secondary structure at 90°C, and has multiple temperature transitions that are less cooperative (the unfolding of the regions is not coordinated). The high beta content, ease of aggregation, high pH and temperature stabilities, and the resistance of aggregates to SDS and GuHCl treatment resembles the behavior of an amyloid protein.;Als5p can form amyloids. The computation program TANGO identified a short sequence (IVIVATT) in the T-region with 93% beta aggregation propensity. A 13mer peptide (SNG-IVIVATT-RTV) of this sequence aggregated into a gel, tested positive for amyloid by increased absorbance when bound to Congo red, increased Thioflavin T fluorescence, and formed amyloid-like fibrils. Purified Ig-T and Ig-T-TR aggregates bound Congo red with increased absorbance and formed amyloid-like fibrils.;Thus in these investigation we show a TR region that is functional by increasing Als5p adherence to ligands and gives conformational flexibility to Als5p. Also we have shown a new property of Als5p to form amyloids and this amyloidicity will increase homotypic (Als5p-Als5p) association and therefore increase cell-cell aggregation.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
