Purification, characterization, and molecular cloning of an outer layer protein of carp fertilization envelope

An outer layer protein of carp fertilization envelope (FE), FEO-1, was puri fied from carp oocytes. The cDNAs encoding FEO-1 were cloned. The mature pr otein of FEO-1 is 21 kDa in molecular weight and contains 177 amino acid re sidues whose sequence has 58% identity to the outer layer protein of chick vitelline membrane. In situ hybridization and immunocytochemistry show that FEO-1 is expressed in oocytes and liver. In oocytes, FEO-1 is stored in th e cortical granules. During cortical reaction, it is exocy-tosed to the per ivitelline space and then gradually added to the outer layer of FE (FEO). F EO-1 first appears as discrete deposits along FEO then merges to form a con tinuous layer. The thickness of FEO increases as cortical reaction proceeds . In addition to FEO-1, FEO contains cystatin, fibroin-like substance (FLS) , and cathepsin-like substance (CLS) as well. They are stored in the cortic al granules and are exocytosed to FEO simultaneously with FEO-1 during cort ical reaction. In FEO, FEO-1 is present in monomer form and can be complete ly extracted by sodium dodecyl sulfate (SDS)mercaptoethanol (MSH). On the o ther hand, the cystatin, FLS, and CLS present in FEO are cross-linked toget her. They are partially extracted by SDS-MSH but can be completely extracte d by guanidium thiocyanate-lauroylsarcosine. (C) 1999 Wiley-Liss, Inc.