STRUCTURAL BASIS FOR DIFFERENCES BETWEEN ELH AND ECG

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The equine gonadotropins, eLH and eCG, bind the same receptors, display both LH and FSH activities, possess identical amino acid sequences, yet differ significantly in their glycosylation patterns. Therefore, all functional differences between them observed in vivo and in vitro are due only to glycosylation. The affinity of eCG for the equine LH receptor is 57- to 75-fold less than that of eLH. The half-life of eCG in circulation is greater than that of eLH. There is widespread use of eCG in biology for promoting follicular development and superovulation either as an end in themselves or as a means to produce large numbers of animals for other studies. Hybrid hormones were prepared from different combinations of intact of ASN56 deglycosylated eLH alpha or eCG alpha preparations with intact eLH beta and e CG beta. Other hybrids consisted of intact alpha- subunit preparations combined with truncated des(121-149)eLH beta or des(121-149)eCG beta preparations. The results of LH and FSH receptor binding assays revealed that alpha Asn56 oligosaccharides and beta- subunit O-linked oligosaccharides exerted a strong negative influence on the receptor binding potency of these hormone preparations. In contrast, alpha Asn82 and beta Asn13 oligosaccharides played a mine role in determining the functional differences between eLH and eCG. Using a variety of modern enzymatic and chemical modifications of oligosaccharides and polypeptide chains of eLH and eCG alpha and beta subunits, a panel of hybrid hormones deficient in specific oligosaccharides will be created. The activities of these hybrids will be tested in a rat testis LH radioligand assay, rat testis Leydig cell steroidogenesis assay in vitro and in an in vivo eCG bioassay. The information obtained will confirm that alpha Asn56 and beta O-linked oligosaccharides have the most significant effect on receptor binding activity and will identify the carbohydrate structures most important for modulating the in vitro steroidogenic and in vivo biological activities of these hormones. Structural studies will identify features of alpha Asn56 and beta O-linked oligosaccharides that are responsible for the differences in bioactivity between eLH and eCG. The results will help to understand the peculiarities of differential glycosylation of proteins in the pituitary and placenta. Because of the unique features of natural glycosylation these studies can not be performed with recombinant DNA technology.