Characterization of D-amino acid-containing neuropeptides from metazoa

Bai, Lu

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Description

Title

Characterization of D-amino acid-containing neuropeptides from metazoa

Author(s)

Bai, Lu

Issue Date

2012-06-27T21:20:28Z

Director of Research (if dissertation) or Advisor (if thesis)

Sweedler, Jonathan V.

Doctoral Committee Chair(s)

Sweedler, Jonathan V.

Committee Member(s)

• Gillette, Rhanor
• Raetzman, Lori T.
• Yau, Peter M.

Department of Study

Molecular & Integrative Physl

Discipline

Molecular & Integrative Physi

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• D-amino acid
• Neuropeptide
• Mass spectrometry
• Liquid chromatography
• Chiral

Abstract

The predominance of left-handedness in amino acids and proteins in living organisms is well established. The presence of D-amino acids in the prokaryotes (e.g., bacterial cell wall and lantibiotics) has been considered exceptions to this rule. In the animal kingdom, proteins are thought to be more strictly built in the L-form due to the chiral selectivity of ribosome-based protein synthesis mechanism. However, in recent years it has become evident that certain residues within a peptide can be inverted to the D-form in an enzyme-mediated post-translational process, forming D-amino acid-containing peptides (DAACPs). DAACPs adopt different three-dimensional structures due to epimerization, and hence, different physicochemical properties and binding affinities to their cognate receptors. These changes have profound consequences on physiological effects. Most of the known DAACPs were found to have greater physiological activities compared to their L-form precursors; and configurational changes also result in resistance to enzymatic digestion, which affects their life cycle and metabolization at the site of action. DAACP Identification is a necessary step toward better understanding of the physiological significance of this modification. More than 30 D-amino acid-containing peptides (DAACPs) have been discovered in metazoan species since the early 1980s. Hormones, antimicrobial peptides and toxins have been found in this list; and in several cases, neuropeptides, a group of cell-to-cell signaling molecules found in the neural tissues, were also shown to undergo such modifications. DAACPs are distributed across different species in several animal phyla, Arthropoda, Mollusca, and Chordata, indicating a wide presence and conservation through evolution. Unfortunately, discoveries of these unusual peptides were oftentimes serendipitous findings that originated from unexpected results from bioassays using the synthetic all-L peptides, and currently there’s no systematic approach to study the presence of these molecules. Conventional peptide sequencing and mass spectrometry-based proteomics approaches do not distinguish L- from D-form residues, either. Therefore, we ask the question: how many more DAACPs have been neglected because of a lack of appropriate analytical methods? How does one differentiate an endogenous neuropeptide—one that contains an amino acid in the D-form—from a complex mixture containing partially characterized brain peptides with concentrations spanning many orders of magnitude? Even without analyzing for DAACPs, this is already a complex measurement challenge. A peptidomics experiment that also seeks to determine D-amino acids present in such samples represents a much more difficult task than quantifying the amino acid configurations of several synthetic peptides, which is the sample type often used when developing characterization approaches. Therefore, methods need to be carefully selected. The development and application of a systematic approach to identify DAACPs from the central nervous systems is presented. Using the classic neurophysiological model organism the sea hare Aplysia californica, a suite of analytical methodologies have been integrated into a discovery funnel that allows identification of DAACPs from complex biological samples. With some modifications, these methods can be applied to a wide range of animal systems including mammals, to test the hypothesis that DAACPs are more common than we currently know.

Graduation Semester

2012-05

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Copyright 2011 Lu Bai

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