Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIR-YFKI, and is identical to the C-terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10(-14) to 10(-9) M for MDF1 and 10(-15) to 10(-5) M for MDF2.