| Leukemia inhibitory factor(LIF) is a multifunctional cytokine, which has been shown to have a wide variety of actions in many different cell types and tissues, including its ability to inhibit HIV-1 replication in vivo and in vitro , preserve the totipotentiality of embryonic stem cells, regulate nerve differentiation, stimulate acute phase protein synthesis by hepatocytes, inhibit lipid accumulation in adipocytes, stimulate the function of osteoblasts and enchance the formation of platelets. In addition, its critical role in female reproduction system have also been reported in recently years, since female mice with LIF-deficient are unresponsive to their viable blastocysts, resulting in implantation failure.LIF exerts its biological actions must through its receptor system,which consists of two distinct membrane-bound glycoproteins, a 190 kD specific LIF-binding receptor α-subunit, gp190, and a 130 kD affinity converter, gp130. LIF binds first to gp190 with low affinity and then to gp130 to form a high affinity functional receptor complex leading to activation of down-stream signal transduction pathways. In addition to the cell membrane-anchored forms of LIF receptor, it has been reported that naturally occurring soluble forms of these receptor molecules are present in biological fluids and may act as natural inhibitors of LIF activity. A soluble form of the mouse gp190 with a molecular weight(Mr) of approximately 90 000-150 000 occurs at high levels in normal mouse serum and is elevated dramatically during pregnancy. It increased sharply around day 8 of pregnancy, just after several days of the transient burst of LIF expression during the implantation process, and peaked at day 12, then dropped to relatively low levels at day 21. An approximately 20-fold increase in the level of transcript encoding soluble LIFR was observed between normal and day 12 pregnant mice. The pattern correlated with the changing levels of soluble mLIFR protein that were detected in the serum.To study the role of the soluble receptor and the mechanism of its production , Tomida et al isolated mouse cDNA for the transmembrane LIFR,and found that an inserted nucleotide sequence introduced a stop codon before the LIFR transmembrane domain, so the mRNA encoded the soluble LIFR α-subunit,sgp190.The recombinant msgp190 containing the extracelluar domain of gp190 by genetic engineering have the same activity with the naturally occurring soluble form of gp190,which further confirmed that sgp190 may be generated by expression of alternatively spliced mRNA of gp190,and the whole or part of gp190 extracellular domain mRNA could encode sgp190 with the identical stimulatory/inhibitory properties of the naturally occurring soluble gp190.Despite the high levels of soluble gp190 appeared in mouse serum, its analogue was not detected in human serum until Zhang et al reported that a soluble form of gp190 was also detected in normal human urine and plasma in 1998. It can inhibit STAT-3 phosphorylation induced by LIF in M1 myeloid cells, which is similar to mouse soluble gp190 function in vitro. But its levels is lower than that in mice serum.This suggested that the soluble human gp190(shgp190) is also produced in the human body and may play some physiological and pathological roles.However,its function remains to be investigated. So we cloned cDNA encoding soluble human gp190, and expressed it in the methylotropic yeast Pichia pastoris. The expression supernatant of GS115/pPIC9K-sgp190 was analysized by SDS-PAGE and western blotting . The results showed that the protein band migrated at MW around 125kD could be specifically recognized by polyclonal antibodies against human sgp190, which indicated the MW of expression protein is about 125kD and have good antigenicity.However,the MW have dramatically difference from its expected MW. We supposed that the higher MW than that estimated from the amino acid sequenced are likely be due to posttranslational modification, including glycosylation.By purification ,we obtaine...
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