Analysis of endogenous d-amino acid-containing peptides in Metazoa

Abstract

Peptides are chiral molecules with their structure determined by the composition and configuration of their amino acid building blocks. The naturally occurring amino acids, except glycine, possess two chiral forms. This allows the formation of multiple peptide diastereomers that have the same sequence. Although living organisms use L-amino acids to make proteins, a group of D-amino acid-containing peptides (DAACPs) has been discovered in animals that have at least one of their residues isomerized to the D-form via an enzyme-catalyzed process. In many cases, the biological functions of these peptides are enhanced due to this structural conversion. These DAACPs are different from those known to occur in bacterial cell wall and antibiotic peptides, the latter of which are synthesized in a ribosome-independent manner. DAACPs have now also been identified in a number of distinct groups throughout the Metazoa. Their serendipitous discovery has often resulted from discrepancies observed in bioassays or in chromatographic behavior between natural peptide fractions and peptides synthesized according to a presumed all-L sequence. Because this L-to-D post-translational modification is subtle and not detectable by most sequence determination approaches, it is reasonable to suspect that many studies have overlooked this change; accordingly, DAACPs may be more prevalent than currently thought. Although diastereomer separation techniques developed with synthetic peptides in recent years have greatly aided in the discovery of natural DAACPs, there is a need for new, more robust methods for naturally complex samples. In this review, a brief history of DAACPs in animals is presented, followed by discussion of a variety of analytical methods that have been used for diastereomeric separation and detection of peptides.

Characterization of GdFFD, a d-Amino Acid-containing Neuropeptide That Functions as an Extrinsic Modulator of the Aplysia Feeding Circuit

“…4 An unusual PTM that is gaining increasing attention is the generation of D-amino acidcontaining peptides (DAACPs) in animals (1). Since the isolation of the first animal peptide having a D-isomerization of an amino acid, the highly analgesic dermorphin from the skin of the South American frog Phyllomedusa (2, 3), more than 30 bioactive DAACPs (including hormones and antimicrobial peptides) have been discovered in frogs (2, 4 -8), mollusks (9 -24), crustaceans (25,26), spiders (27,28), and even mammals (29,30).…”

“…More-over, methods that are compatible with small quantities of tissue samples are desirable as neuropeptide studies oftentimes are sample limited. Second, although DAACPs have been shown to be more bioactive in the CNS in several animals (1,34), it is not known how D-amino acid-containing neuropeptides might ultimately act on a specific neural circuit to affect network output and behavior. In fact, to the best of our knowledge, no studies have examined the effects of DAACPs on a defined neural circuit, likely due to the limited information on well defined neural circuits in most species.…”

“…Like other PTMs, identification of DAACPs requires the use of biochemical and/or analytical chemical methods (1). However, this PTM is challenging to detect as it does not change the elemental composition, molecular formula, or even the mass of the peptide.…”

“…However, this PTM is challenging to detect as it does not change the elemental composition, molecular formula, or even the mass of the peptide. Diastereomer separation techniques such as chromatography and capillary electrophoresis have been developed using synthetic peptides and used reliably to confirm the presence of DAACPs in various samples (1). Recently, we showed that the fragmentation pattern generated during MS/MS sequencing can be useful for distinguishing neuropeptide epimers in identified neurons known to express candidate DAACP prohormones (38).…”

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

“…Since several decades, numerous protocols have been described in the literature, which include derivatization of amino acids with chiral reagent followed by chromatography (review in [17]). This approach, though technically not very straightforward and which needs nanomolar amounts of peptide (review in [18]), still remains the most accurate and reliable method to determine the stereochemistry of AA residues in peptides.…”