Post-translational Processing of the Insulin-like Growth Factor-2 Precursor : ANALYSIS OFO-GLYCOSYLATION AND ENDOPROTEOLYSIS

We are studying mice that carry a targeted disruption of the gene encoding insulin-like growth factor II (IGF-II). Transmission of this mutation through the male germline results in heterozygous progeny that are growth deficient. In contrast, when the disrupted gene is transmitted maternally, the heterozygous offspring are phenotypically normal. Therefore, the difference in growth phenotypes depends on the type of gamete contributing the mutated allele. Homozygous mutants are indistinguishable in appearance from growth-deficient heterozygous siblings. Nuclease protection and in situ hybridization analyses of the transcripts from the wild-type and mutated alleles indicate that only the paternal allele is expressed in embryos, while the maternal allele is silent. An exception is the choroid plexus and leptomeninges, where both alleles are transcriptionally active. These results demonstrate that IGF-II is indispensable for normal embryonic growth and that the IGF-II gene is subject to tissue-specific parental imprinting.

Growth factors are thought to function as pivotal autocrine-paracrine regulatory signals during embryonic development. Insulin-like growth factor II (IGF-II), a mitogenic polypeptide for a variety of cell lines, could have such a role, as indicated by the pattern of expression of its gene during rodent development. The IGF-II gene uses at least three promoters and expresses several transcripts in many tissues during the embryonic and neonatal periods, whereas expression in adult animals is confined to the choroid plexus and the leptomeninges. To examine the developmental role of IGF-II, we have begun to study the consequences of introducing mutations at the IGF-II gene locus in the mouse germ line. We have disrupted one of the IGF-II alleles in cultured mouse embryonic stem (ES) cells by gene targeting and constructed chimaeric animals. Germ-line transmission of the inactivated IGF-II gene from male chimaeras yielded heterozygous progeny that were smaller than their ES cell-derived wild-type littermates (about 60% of normal body weight). These growth-deficient animals were otherwise apparently normal and fertile. The effect of the mutation was exerted during the embryonic period. These results provide the first direct evidence for a physiological role of IGF-II in embryonic growth.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.