Rat complement factor I: molecular cloning, sequencing and expression in tissues and isolated cells

Factor I (Fn is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b thereby inactivating these proteins. The human protein and the recently characterized mouse factor I are heterodimers of about 88 000 MW which consist of a non-catalytic heavy chain of 50 000 MW which is l inked to a catalytic light chain of 38 000 MW by a disulphide bond. For the screening of a rat liver cDNA library we used a hybridization probe produc ed by polymerase chain reaction (PCR) using degenerated primers which corre sponded to conserved parts of the human and the murine factor I nucleotide sequences. One of the identified sequences, which had a length of 2243 base pairs (bp), contained the complete coding region and the whole 3' untransl ated region. The length of the coding region in rat consisted of 1812 bp fo llowed by a 3' untranslated region of 207 bp including the polyadenylation signal and the beginning of the poly A tail. Comparison of the rat cDNA-der ived coding sequence revealed identities of 87% to the mouse and of 78% to the human FI nucleotide sequence. The translation product of rat FI mRNA wa s 604 amino acid residues (aa) in length with an identity of 85% to the mou se (603 aa) and 69% to the human protein (583 aa). The comparison of the mo lecular mass predicted by the primary structure and derived from rat FI iso lated from rat serum as detected in immunoblot analyses suggested a glycosy lation of more than 20% of the total mass of the FI protein. Expression stu dies using reverse transcription (RT)-PCR assays indicated that FI-specific mRNA could neither be identified in B cells, nor in T cells, monocytes or granulocytes from rat and human peripheral blood nor in rat peritoneal macr ophages. These data were in agreement with the results of RT-PCR obtained w ith several human lymphoma cell lines (Jurkat, MOLT-4, HUT102 Wil 2-NS, Ram os, Raji, U937) all of which were devoid of FI-specific mRNA. In accord wit h our data from two rat hepatoma cell, lines (FAO and H4IIE) and one from m an (HepG2) only isolated rat hepatocytes (HC) but neither Kupffer cells (KC ), hepatic stellate cells (HSC; Ito cells) nor sinusoidal endothelial cells (SEC) expressed FI-specific mRNA. FI mRNA was also detected in human umbil ical vein endothelial cells (HUVEC) and in the uterus and small intestine o f the rat. Spleen and lymph nodes did not contain any detectable FI-specifi c mRNA.