Biomedical subjects
Publications and source records attributed to C Estaller.
Using PCR and Northern blot analysis, an IL-1 receptor antagonist specific transcript was amplified from HepG2- and liver mRNA. cDNA clones coding for IL-1 receptor antagonist were isolated from a liver cDNA library and sequence comparison revealed complete identity with the secreted, monocytic form of IL-1 receptor antagonist.
The nucleotide and complete amino acid sequence for the human beta 2-glycoprotein I (beta 2I) was derived by sequencing the cDNA clone pB2I-1. In addition to the 326 amino acid residues of the mature protein this clone codes for a putative leader peptide and contains sequence representing 5' and 3' untranslated regions. When this amino acid sequence was compared with the previously published primary sequence, three major amino acid substitutions were found, two involving cysteine residues. These substitutions lead to a new alignment of the complement control protein (CCP) repeats present in beta 2I and a prediction of the complete disulphide bond organization. Northern-blot analysis indicates that hepatocytes are a major site of biosynthesis for this protein. A transcription signal of about 1.5 kb was detected by using RNA from HepG2 cells.
Three factor H mRNA species of 4.3 kb, 1.8 kb, and 1.4 kb are constitutively expressed in human liver. Having previously characterized full-length cDNA clones derived from the 4.3-kb and 1.8-kb factor mRNA, we report here the isolation and eucaryotic expression of full-length cDNA clones coding for the 1.4-kb mRNA species. The 1266-bp cDNA codes for a polypeptide of 330 amino acids and contains two polyadenylation signals and a short poly(A)+tail. The protein is composed of a leader peptide followed by five short consensus repeat domains. It shows a hybrid structure with the last three domains being almost identical to the carboxy-terminal of the classical 150-kDa factor H molecule and the two first domains representing unique short consensus repeat structures. Eucaryotic expression in COS7 cells revealed two polypeptides derived from one cDNA clone that are also found in human serum. Differences between the cDNA clones within the last three domains indicate two distinct, possibly allelic sequences that, in addition, differ from the authentic 150-kDa factor H sequence. Southern blot results support the notion that the 4.3-kb factor H and the 1.4-kb factor H-related mRNA are transcribed from two separate but highly homologous genes.
The human complement factor H is an important component in the control of the alternative pathway of complement activation. We have previously shown that a least three factor H homologous mRNA species of 4.3 kb, 1.8 kb and 1.4 kb in length are constitutively expressed in human liver. In addition, several factor H-related proteins have been detected in human sera using antibodies directed against the classical human factor H glycoprotein of 150 kDa. The structure of the additional polypeptides has not been shown so far. Circumstantial evidence suggests that the 1.8-kb mRNA might encode the 43-kDa factor H-like polypeptide. Here we report the isolation, characterization and eukaryotic expression of the first full-length cDNA representing the major 4.3-kb mRNA and the 1.8-kb mRNA of human factor H. We show that the 4.3-kb transcript encodes the 150-kDa-factor H glycoprotein and the 1.8-kb mRNA the 43-kDa factor H polypeptide. The identity of the two cDNA in a region of 1400 nucleotides suggests that the two factor H-related transcripts are derived from one gene by a process of alternative splicing.
Previously, we have shown that three different mRNA species of 4.3 kb, 1.8 kb and 1.4 kb, related to human complement factor H, are constitutively expressed in the human liver. Probing with our cDNA clone H-46 which represents 920 bp of the 3' end of the 4.3 kb mRNA of factor H on human liver RNA, we always detected the 4.3 kb and the additional, abundantly expressed mRNA species of 1.4 kb in length, indicating that the 1.4 kb transcript is highly homologous to the 3' end of the classical factor H mRNA of 4.3 kb. Using H-46 as a probe, several cDNA clones were isolated from a liver cDNA library and sequenced. The open reading frame of the novel mRNA species encodes a peptide consisting of five internal short consensus repeat motifs (SCR), identifying the translational product to be a member of the SCR family. Sequence comparison with cDNA clones derived from liver RNA of a different donor provided evidence for variability in the factor H related proteins encoded by the 1.4 kb mRNA species. Interestingly, this variability was found to be restricted to the three carboxyterminal SCR domains. Expression data indicate that our variant is not recognized by the monoclonal antibody 3D11.