Intracellular Degradation of C-Peptides in Molluscan Neurons Producing Insulin-Related Hormones

Li, K.W.; Jiménez, Connie R.; Vonk, N.; Dreisewerd, Klaus; Hillenkamp, Franz; Karas, Michael; Geraerts, W.P.M.

doi:10.1016/s0196-9781(97)00020-x

Cited by 14 publications

(12 citation statements)

References 29 publications

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“…As further evidence that the Leu-Leu processing is not caused by extracellular degradation, the atrial gland forms were observed even when atrial glands were boiled before extraction (20). In the bag cells, the doubly cleaved peptide (ELH [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] ) is observed. Although these shortened forms of ELH have not been identified or sequenced previously, shortened ELH forms have been postulated based on immunoassay results (10,33).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies with Lymnaea have demonstrated that the acidity (ϷpH 2) of the DHB matrix, combined with short sample handling times, is likely to inactivate endogenous proteolytic enzymes (21,22). However, because gradual peptide degradation is reported to occur in bag cell cluster extracts, even when incubated in 0.2 M HCl for 2-3 hr (29), we investigated a number of other sample preparation paradigms.…”

Section: Peptides In Bag Cellmentioning

confidence: 99%

“…When combined with knowledge of the prohormone, identification of most, if not all, of the peptides present in complex biological samples is possible. Previous studies have used MALDI to study gene expression within identified neurons from the pulmonate snail Lymnaea stagnalis (21,22). Additional MALDI-based methods detect novel in vitro processing of neuropeptide Y (NPY) in human cerebrospinal fluid (23) and monitor peptide changes in the neurointermediate lobe of rats (24).…”

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confidence: 99%

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Proteolytic processing of the Aplysia egg-laying hormone prohormone

Garden

Shippy

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

114

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By using matrix-assisted laser desorption͞ ionization time-of-f light MS, individual peptidergic neurons from Aplysia are assayed. A semiquantitative method is developed for comparing single-cell profiles by using spectral normalization, and peptides are localized to specific cells by mass spectrometric cell mapping. In addition to all previously identified products of the egg-laying hormone (ELH) gene, other peptides are formed from proteolytic hydrolysis of Leu-Leu residues within ELH and acidic peptide (AP). AP exhibits further processing to yield AP 1-20 and AP 9 -27 . These peptides appear to be colocalized in vesicles with ELH, transported to specific neuronal targets, and released in a Ca 2؉ -dependent manner. A differential peptide distribution is observed at a specific target cell, and a low-frequency variation of AP, [Thr 21 ]AP, is detected in a single animal.

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Section: Discussionmentioning

confidence: 99%

Section: Peptides In Bag Cellmentioning

confidence: 99%

mentioning

confidence: 99%

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Proteolytic processing of the Aplysia egg-laying hormone prohormone

Garden

Shippy

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

114

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“…In several cases in gastropod molluscs, such peptides are rapidly degraded, and some of these intermediates can be detected with MALDI MS (de With et al, 1997;Garden et al, 1998). Several of these linker peptides predicted by the prohormone were also detected in the spectra (data not shown).…”

Section: Processing Of the Amrp Precursormentioning

confidence: 96%

TheAplysia MytilusInhibitory Peptide-Related Peptides: Identification, Cloning, Processing, Distribution, and Action

et al. 1999

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Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.

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“…In such a procedure, detailed molecular weight profiles of single-cell contents could be obtained without intermediate steps, thereby revealing information previously buried in statistical averages. The work by Sweedler et al (1) exemplifies the power of such a single-cell molecular weight profiling tool (15)(16)(17)(18).…”

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confidence: 99%