Identification of Neuropeptides from the Sinus Gland of the Crayfish Orconectes limosus Using Nanoscale On-line Liquid Chromatography Tandem Mass Spectrometry

Abstract

In this article, a novel and sensitive analytical strategy for direct characterization of neuropeptides from the X-Organ-sinus gland neurosecretory system of the crayfish Orconectes limosus is presented. A desalted extract corresponding to 0.5 sinus gland equivalents was analyzed in a nanoflow liquid chromatography system coupled to quadrupole time-of-flight tandem mass spectrometry (nanoLC-QTOF MS/MS). The existence and structural identity of four crustacean hyperglycemic hormone precursor-related peptide variants and two new genetic variants of the pigment-dispersing hormone, not detected by conventional chromatographic systems, molecular cloning, or immunochemical methods before, was revealed. Studies in crustaceans have demonstrated the X-Organsinus gland neurosecretory system (XO-SG) 1 as a source of various neuropeptides (1, 2). The most relevant progress has been achieved in structure elucidation of a novel family of large peptides from the sinus gland (SG), which includes, according to their first-discovered biological activities, crustacean hyperglycaemic (CHH), moult-inhibiting (MIH) and vitellogenesis/gonad-inhibiting (VIH/GIH) hormones (3, 4). Two subfamilies may be distinguished at the preprohormone level, either containing an associated CHH precursor-related peptide sequence (CPRP), located between the signal peptide and the native hormone sequence, characteristic for the CHH subfamily, or without this CPRP sequence, as determined for the MIH/VIH/GIH subfamily. CPRPs have also been identified in other invertebrate groups such as insects and nematodes (4, 5). These neurohormones are classically considered as major physiological regulators involved in the control of carbohydrate metabolism and in various functions in development such as moult and reproduction (3, 6, 7). Additionally, the chromatophore-regulating hormones, like red pigmentconcentrating hormone (RPCH) and pigment-dispersing hormone (PDH), have been found in the XO-SG system of different crustacean species (8 -11).Different approaches, such as conventional chromatography, molecular cloning, and immunochemical methods, were used previously to investigate sinus gland components (12)(13)(14)(15)(16)(17). The limitations of these established methods to identify and characterize new peptides are the relatively low sensitivity in the case of conventional chromatography, the necessity of sufficient sequence homology in the case of molecular cloning, and the availability of specific antibodies in the case of immunochemical methods. In contrast to this, the combination of nanoscale liquid chromatography and mass spectrometry (MS) capable of tandem MS analysis (nanoLC-MS/MS) is designed for detection and sequencing of known and unknown peptides independent of their sequence homology. This is the first report on the direct analysis of neuropeptides from the neurosecretory XO-SG system using nanoLC-MS/MS.

Circadian clocks in crustaceans: identified neuronal and cellular systems

“…These are neurons with T-shaped branches that, together with several other PDH-ir cell groups, form a prominent tract system predominantly in the medulla terminalis, which also contains axons connecting to the brain and to the contralateral eyestalks (arrows in Figure 4A,B) (138,140,141). Since the origins of up to three different identified PDH-isoforms in sinus glands are still not clear (140,211), this problem presents a further complication in the analysis of all these groups of PDH-neurons and for the analysis of possible physiological effects of these hormonally released PDH-isoforms. There is also evidence that these PDH-isoforms are differentially distributed in visual eyestalk ganglia (e.g.…”

“…However, notably, for hatching larvae released from intertidal or DVM-performing crabs circatidal rhythm components are certainly more important (193)(194)(195). detected in the crabs Callinectes sapidus (206), Cancer borealis (207,208) and Cancer productus (140), the shrimps Litopenaeus japonicus (209) and Litopenaeus vannamei (210), the crayfish Orconectes limosus (211), and the lobster H. americanus (207). In the CNS of diverse crustacean species, multiple neurosecretory and interneurons express PDHs (137,138,140,141,152), but innervation of the sinus gland as known e.g.…”

The neuroendocrine system of invertebrates: a developmental and evolutionary perspective

“…Two other neurohemal structures, called the postcommissural and the pericardial organs, receive projections from NSCs in the brain and ventral nerve cord. A large variety of neuropeptides influencing pigmentation, carbohydrate levels, osmoregulation, growth/molting, and reproduction are released from each of these sites (Bulau et al 2004, Serrano et al 2004.…”

A novel form of pigment‐dispersing hormone in the central nervous system of the intertidal marine isopod, Eurydice pulchra (leach)

“…Asterisk indicates dorsal arborizing dendrites (see D for orientation). Scale bar ¼ 100 lm in A; 50 lm in D-H; 25 lm in F,G; 20 lm in B and inset to C. Klein et al, 1994;Desmoucelles-Carette et al, 1996;Yang et al, 1999;Bulau et al, 2004). Notably, a recent report (Hsu et al, 2008) has convincingly demonstrated a differential distribution of b-PDH-I and -II isoforms in the eyestalk of Cancer productus, indicating that the former is likely to function as a neurotransmitter/modulator, whereas the latter functions as a neurohormone, and is only expressed by neurons that terminate in the SG.…”