Abstract
The rainbow trout egg vitelline envelope (VE) is constructed of three proteins, called VEalpha,VEbeta, and VEgamma, that are synthesized and secreted by the liver and transported in the bloodstream to the ovary, the site of VE assembly around eggs. All three proteins possess an N-terminal signal peptide, a zona pellucida domain, a consensus furin-like cleavage site (CFLCS) close to the C terminus, and a short propeptide downstream of the CFLCS. Proteolytic processing at the CFLCS results in loss of the short C-terminal propeptide from precursor proteins and enables incorporation of mature proteins into the VE. Here mass spectrometry (matrix-assisted laser desorption ionization time-of-flight-mass spectrometry and liquid chromatography-mass spectrometry with a micromass-quadrupole TOF hybrid mass and a QSTAR Pulsar i mass spectrometer) was employed with VE proteins isolated from rainbow trout eggs in a peptidomics-based approach to determine the following: 1) the C-terminal amino acid of mature, proteolytically processed VE proteins; 2) the cellular site of proteolytic processing at the CFLCS of VE precursor proteins; and 3) the relationship between proteolytic processing and limited covalent cross-linking of VE proteins. Peptides derived from the C-terminal region were found for all three VE proteins isolated from eggs, indicating that processing at the CFLCS occurs after the arrival of VE precursor proteins at the egg. Consistent with this conclusion, peptides containing an intact CFLCS were also found for all three VE proteins isolated from eggs. Furthermore, peptides derived from the C-terminal propeptides of VE protein heterodimers VEalpha-VEgamma and VEbeta-VEgamma were found, suggesting that a small amount of VE protein can be covalently cross-linked on eggs prior to proteolytic processing at the CFLCS. Collectively, these results provide important evidence about the process of VE formation in rainbow trout and other non-cyprinoid fish and allow comparisons to be made with the process of zona pellucida formation in mammals.
Highlights
Introduction to Mass Spectrometry Analyses ofRainbow Trout VE Proteins—It is to be expected that the bulk of VE protein associated with rainbow trout eggs is mature, processed, and assembled, with only a relatively small fraction present as immature, unprocessed VE proprotein
The presence of the C-terminal propeptide in VE was confirmed by other MALDI
By using LC-MS(QSTAR) another peptide was detected from the C-terminal propeptide of VE (Fig. 3B)
Summary
Introduction to Mass Spectrometry Analyses ofRainbow Trout VE Proteins—It is to be expected that the bulk of VE protein associated with rainbow trout eggs is mature, processed, and assembled, with only a relatively small fraction present as immature, unprocessed VE proprotein. MS was used to identify the C-terminal amino acid of VE proteins present on rainbow trout eggs and to determine whether the C-terminal propeptide, downstream of the CFLCS, as well as an intact CFLCS, are present on at least a portion of the VE proteins surrounding eggs. To evaluate the mass spectra that follow
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