Elsevier

Journal of Chromatography B

Volume 879, Issue 29, 1 November 2011, Pages 3102-3107
Journal of Chromatography B

Review
Experimental strategies for the analysis of d-amino acid containing peptides in crustaceans: A review

https://doi.org/10.1016/j.jchromb.2011.03.032Get rights and content

Abstract

Detection of d-amino acids in natural peptides has been, and remains a challenging task, as peptidyl isomerization is a peculiar and subtle posttranslational modification that does not induce any change in primary sequence or in physicochemical properties of the molecule such as molecular mass or pI. Therefore, the presence of a d-amino acid residue in a peptide chain is generally transparent to classical methods of peptide analysis (electrophoresis, chromatography, mass spectrometry, molecular biology). In this article, we will review the various experimental strategies and analytical techniques, which have been used to characterize and to study d-amino acid containing peptides in crustaceans.

Introduction

An old dogma in biology is that proteins are composed of amino acids in the l-configuration exclusively. However in the late 30s, the presence of peptidyl poly-d-glutamic acid in the bacterial cell envelope of the virulent Bacillus anthracis was demonstrated [1], and, in the 40s, a rapidly increasing number of microbial peptide antibiotics containing frequently unusual amino acid residues and notably d-amino acids (d-AAs) were discovered (review in [2]). Natural occurrence of d-AAs in proteins was rather considered as a peculiarity of microorganisms. However, d-AA residues were later found in proteins from animals, including Man. For example, d-Asp has been found in several human proteins, such as dentin [3], α-crystalline from lens of patients with cataract [4], and β-amyloid peptide from brains of Alzheimer's patients [5]. In these long-living proteins, the origin of peptidyl d-AAs may be explained by non-enzymatic racemization and isomerization associated with aging or diseases [6].

In another context, a d-Ala residue was reported to be present in an opioid peptide from skin secretion of the tree frog Phyllomedusa sauvagei [7]. At the time of publication, the scientific community was quite doubtful about this work, which was yet a real breakthrough in peptide studies. Since, d-AA residues of different nature have been found in bioactive peptides from venom or nervous tissue from various species belonging to molluscs, arachnids, crustaceans and vertebrates (platypus and frogs) (review in [8]). Only one d-AA was found in the peptide chain, and close to one end, most frequently the N-terminus. Moreover, a classical codon has always been found in the mRNA in the position where the d-residue is present in the mature peptide [9].

Several hypotheses may be formulated to explain the presence of a d-residue in the peptide chain. It may result from different mechanisms such as conversion of a free l-AA into its d-counterpart before its incorporation, or enzymatic posttranslational modification of an l-residue after the peptide chain synthesis. This is the case of the crustacean neuropeptides that have been studied in our laboratory for many years. Indeed, Crustacean Hyperglycaemic Hormone (CHH) and Vitellogenesis Inhibiting Hormone (VIH) are 70- to 80-residue neuropeptides, depending of the peptide and of the considered species. They exhibit hormonal activities and regulate energetic metabolism and reproduction, respectively. CHH constitutes the archetype of a peptide family present in Arthropods. This family is mostly characterized by six cysteyl residues at conserved positions, paired in three-disulfide bridges (see Table 1). CHH and VIH were shown to be present in the major neuroendocrine organ of the lobster Homarus americanus, the X organ-sinus gland complex, as two isomers differing by the change of a specific residue from the l- to the d-configuration. This change results in modifications of the biological activity of the peptide [10].

In the course of studies of these peptides, a number of different analytical methods have been utilized and the following text describes the strategies developed. To conclude, focus will be on an approach seldom made in this field, which is the study of d-amino acid containing peptides (DAACPs) at the cellular level, by immunohisto- and immuncytochemistry.

Section snippets

Different hydrophobicity in RP-HPLC for l- and d-amino acid containing peptides from crustaceans: peptide mapping and chiral amino acid analysis

As numerous discoveries in Life Sciences, the discovery of DAACPs in crustaceans has relied on the anecdotic observation, in the early 90s, that CHH and VIH from neuroendocrine glands of the lobster Homarus americanus were eluted from C-18 RP-HPLC (reverse phase – high performance liquid chromatography) columns as pairs of peaks composed of peptides with the same molecular mass, pI, amino acid composition and N-terminal sequence [11], [12], [13] (Fig. 1). It was then proposed that these

Identical N-terminal sequences for DAACPs of different species: RP-HPLC and ELISA with specific antibodies

Once the nature of the d-residue in CHH has been established in the American lobster by the analytical methods described above, it was decided to design specific tools, namely specific antibodies, to search for similar peptides in other species and to study the production site in crayfishes and lobsters.

For that sake, conformational antibodies have been developed against synthetic peptides with sequences corresponding to the N-terminal octapeptides of l- and DPhe3 CHH. Synthetic peptides, oct-L

Peptide mapping, comparison with synthetic peptides and validation with specific antibodies: the example of VIHs

In DAACPs characterized in animals so far, the d-residue is found most frequently near the N-terminus (until residue 4). A few years ago, a new approach founded on this peculiarity was developed to study VIH isoforms of the American lobster. The stereoisomers (named VIH I and VIH II since 1991) were characterized by an approach combining complementary techniques of bio- and immuno-chemistry [21].

In a first step, retention times in RP-HPLC were compared between (i) a panel of synthetic

d-Amino acid detection and quantification in crustacean peptides by mass spectrometry

In initial studies described above, mass spectrometry (MS) has been utilized as an analytical tool to determine the mass of neuropeptides or fragments, the detection of d-residues relying on chiral amino acid analysis or/and recognition by specific conformational antibodies (Table 2). However, MS has been applied for several years to differentiate all-l peptides from those containing a d-residue. This was made possible because the presence of a d-residue induces a difference in the

d-Amino acid containing peptides detection by immunohisto/cyto-chemistry

As described above, immunoassays (ELISA) using specific and polyclonal antibodies against the N-terminus of CHH and VIH stereoisomers have allowed detecting DAACPs in various crustacean species. Otherwise, DAACPs quantification by a combination of indirect ELISA (double sandwich) and RP-HPLC was described in [27].

In the literature, besides works on crustacean peptides detailed below, a very few studies of DAACPs at the cellular and subcellular levels have been reported. Two of them dealt with

Conclusion – futures investigations

To conclude, other analytical approaches may be used to identify and to analyze DAACPs. For example, Nuclear Magnetic Resonance (NMR) experiments have been performed to confirm the presence of a d-residue in two platypus toxins, C-type natriuretic peptide (d-Leu2 for OvCNPb) and defensin-like peptide (d-Met2 for DLP-2) [34], [35]. In NMR experiments on the conotoxin peptide, conomorphin, it was demonstrated the DPhe13 is necessary to form a tight loop in the middle of the peptide, as this loop

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    This paper is part of the special issue “Analysis and Biological Relevance of d-Amino Acids and Related Compounds”, Kenji Hamase (Guest Editor).

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