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J. Biol. Chem., Vol. 280, Issue 45, 37585-37598, November 11, 2005

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Mass Spectrometric Evidence That Proteolytic Processing of Rainbow Trout Egg Vitelline Envelope Proteins Takes Place on the Egg^*

Costel C. Darie, Martin L. Biniossek, Mary A. Gawinowicz¶, Yelena Milgrom¶, Joerg O. Thumfart, Luca Jovine, Eveline S. Litscher, and Paul M. Wassarman1

From the Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, New York 10029-6574, Institut fuer Molekulare Medizin und Zellforschung/Physiologie II, Albert-Ludwigs Universitaet, Stefan-Meier-Strasse 19, 79104 Freiburg, Germany, ^¶Protein Core Facility, Columbia University College of Physicians and Surgeons, New York, New York 10032-3702

The rainbow trout egg vitelline envelope (VE) is constructed of three proteins, called VE,VE, and VE, 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 VE-VE and VE-VE 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.

Received for publication, June 20, 2005 , and in revised form, August 24, 2005.

^* This work was supported in part by National Institutes of Health Grant HD-35105 (to P. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Brookdale Dept. of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574. Tel.: 212-241-8616; Fax: 212-860-9279; E-mail: paul.wassarman{at}mssm.edu.

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