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DNA sequence and rflp analysis for the definition of human complement c4 polymorphism based on isotype-specific pcr amplification
M O L E C U L A R C H A R A C T E R I Z A T I O N OF T H E C 8 ~ - m R N A FROM A C 8 ~ - D E F I C I E N T FAMILY. Oscar Burrone*, Sabrina Mancardi* and Francesco Tedesco#. *, International Centre for Genetic Engineering and Biotechnology, ICGEB-UNIDO, Trieste, Italy and #, Istituto di Patologia Generale, Universit& di Trieste, Trieste, Italy. The inherited C8 deficiency occurs more frequently as a defect of the production of the C813 subunit. We have approached the study of a family case with C813 deficiency by the molecular analysis of the mRNA produced by PBMC. The family consisted of two siblings lacking immunochemically detectable serum C8f3 subunit, and the two heterozygous parents. The analysis was carried out by RT/PCR of the C813mRNA, using a set of appropriate primers, and followed by cloning and sequencing of the obtained fragments. A deletion of 241 nucleotides, from nt. 865 to nt 1105 (with respect to the ATG initiation codon), was detected in the mRNA of the two siblings. This defect produces a frameshift with the consequence of creating a new stop codon at position 1199. The internally deleted mRNA would code for a truncated C8~ subunit, containing 31 new additional amino acids in its COOH end. This abnormality, compatible with an alteration at the level of splicing of the C8~-RNA precursor molecule, has so far been detected in the two siblings and in the mother. Studies are in progress to define the nature of the paternal defect in the C8!3 gene.
Elevated
complement with HDL deficiency p r e s e n c e of normal level SP-40,40 ) and the atherosclerosis
(
activities of the patients Tangier disease ) : The of clusterin ( a p o l i p o p r o t e i n J, possible implication in the
Nam-Ho Choi-Miural), Terufumi Sakamotol}, Yasuko Nakanol), Takashi Tobel), Motowo Tomita~}, Shuji Ishizawa2), Yasushi Takagi2), Kunihide Gomi~), Haruki Nakarnura3), Nobuaki Kida4) and Sachiya Ohtaki4) ~)Dept. Physiol. Chem., Sch. Pharm Sci., 2)Dept. Clin. Pathol., Sch. Med., Showa University, 3)Dept. Meal., Hamanomachi General Hospital and 4)Dept. Lab. Med., Miyazaki Medical College Clusterin ( apolipoprotein J, SP-40,40 ) is a muitifunctional protein which modulates the membrane attack complex formation of complement, causes cell aggregation, accelerates the immune complex formation, binds to ~endorphin and stabilizes plasminogen activator inhibitor type 1. Clusterin formes a minor subclass of high density lipoprotein ( HDL ) with apolipoprotein A-I ( apo A-I ) in serum. Recently, we reported that the HDL consisting of ctusterin, apo A-I and lipids was incorporated into the soluble membrane attack complex ( SMAC ) of complement at the stage of SC5b-7 via clusterin as a binding site. Clusterin is an apolipoprotein which is present only in HDL but not in low density Upoprotein ( LDL ) as well as apo A-I and apolipoprotein All ( apo A-II ). In spite of the deficiencies of apo A-I, apo A-II and HDL in the sera of the patients with Tangier disease, clusterin was found in them at normal level. While clusterin was present as the component of HDL with apo A-I in the sera of normal donors, it was present as a protein which did not form a complex in the sera of Tangier patients. SC5b-9 made from the sera of Tangier patients contained normal amount of clusterin and was deficient in apo A-I, indicating that clusterin could be incorporated into SMAC without apo A-I. The complement activities of the sera of the patients were higher than those of normal donors. These results may be explained by the deficiencies of apo A-I, apo A-II and HDL in the patients because they were suggested to be the inhibitors of the reactive hemolySis of complement. The elevated complement activities of the patients might be related to the severe atheroselerotic lesions in Tangier disease.
DNA SEQUENCE AND RFLP ANALYSIS FOR THE DEFINITION OF HUMAN COMPLEMENT C4 POLYMORPHISM BASED ON ISOTYPE-SPECIFIC PCR AMPLIFICATION X. Chu, G. Barba, L. Braun-Heimer, C. Rittner, K. Witzel, and P.M. Schneider Institute of Legal Medicine, Johannes Gutenberg-University, Mainz, Germany. The fourth component of the complement system, C4, is encoded by two highly similar, HLA-linked genes, expressing the two different isotypes C4A and C4B. A two-step "nestedPCR" approach was developed to facilitate the isotype-specific amplification of selected exons of the C4 genes. Using PCR primers directed at the sequences coding for residues 11011106 (Chido 4 serological determinant) capable of distinguishing the isotype-specific C4B sequence in the C4d region, in combination with primers from different exon-flanking sequences of the C4 genes, DNA fragments covering exons 10 to 41 from several C4B alleles were amplified. These 5-7 kb long isotype-specific PCR products were then subjected to a second PCR amplification generating fragments of 1-2 kb size suitable for direct DNA sequencing and RFLP analysis. Thus a number of C4B alleles have been sequenced to define the genetic basis of polymorphism. Based on these findings either allele-specific PCR or RFLP analysis of PCR products can be used for the rapid typing of the C4 alleles. [Supported by DFG (Schn 284/3-1 )]
GENERATION OF A FUNCTIONAL DOMAIN OF CRI WITH INCREASED LIGAND BINDING A N D COFACTOR ACTIVITIES. Liliana Clemenza, Richard Hauhart, John Atkinson, Malgorzata Krych. Washington Univ. Sch. of Med., St., Louis, MO 63110, USA There are two distinct binding/active sites in CRI; SITE 1 with short consensus repeats (SCRs) -1/2 and SITE 2 with SCRs-8/9. C4b is a major ligand/substrate for SITE 1. Both C3b and C4b are ligands/substrates for SITE 2. In SITE 1, two conserved amino acids (aa), G in SCR-1 and Y in SCR-2, as well as two nonconserved aa in SCR-2, R and N, were important for C4b binding. Conserved aa are likely a part of a framework of a SCR, whereas nonconserved aa may be involved in direct interactions. We now report that, when these four aa important for C4b binding in SITE 1, were transferred to the homologous positions of SITE 2, functional activity of SITE 2 was increased for both ligands/substrates. As assessed by ELISA over a wide range of ionic strengths, protein carrying this modified SITE 2 bound iC3/C3b and C4b better than the protein with wild type SITE 2. Moreover, it had increased cofactor activity for factor I cleavage of both C3b and C4b. Thus, by genetic manipulation of SCRs, we have constructed a regulator of complement activation (RCA) protein, in this case derived from CRI, with increased functional activity. More effective inhibitors of complement activation can be produced by this strategy.
Elevated
complement with HDL deficiency p r e s e n c e of normal level SP-40,40 ) and the atherosclerosis
(
activities of the patients Tangier disease ) : The of clusterin ( a p o l i p o p r o t e i n J, possible implication in the
Nam-Ho Choi-Miural), Terufumi Sakamotol}, Yasuko Nakanol), Takashi Tobel), Motowo Tomita~}, Shuji Ishizawa2), Yasushi Takagi2), Kunihide Gomi~), Haruki Nakarnura3), Nobuaki Kida4) and Sachiya Ohtaki4) ~)Dept. Physiol. Chem., Sch. Pharm Sci., 2)Dept. Clin. Pathol., Sch. Med., Showa University, 3)Dept. Meal., Hamanomachi General Hospital and 4)Dept. Lab. Med., Miyazaki Medical College Clusterin ( apolipoprotein J, SP-40,40 ) is a muitifunctional protein which modulates the membrane attack complex formation of complement, causes cell aggregation, accelerates the immune complex formation, binds to ~endorphin and stabilizes plasminogen activator inhibitor type 1. Clusterin formes a minor subclass of high density lipoprotein ( HDL ) with apolipoprotein A-I ( apo A-I ) in serum. Recently, we reported that the HDL consisting of ctusterin, apo A-I and lipids was incorporated into the soluble membrane attack complex ( SMAC ) of complement at the stage of SC5b-7 via clusterin as a binding site. Clusterin is an apolipoprotein which is present only in HDL but not in low density Upoprotein ( LDL ) as well as apo A-I and apolipoprotein All ( apo A-II ). In spite of the deficiencies of apo A-I, apo A-II and HDL in the sera of the patients with Tangier disease, clusterin was found in them at normal level. While clusterin was present as the component of HDL with apo A-I in the sera of normal donors, it was present as a protein which did not form a complex in the sera of Tangier patients. SC5b-9 made from the sera of Tangier patients contained normal amount of clusterin and was deficient in apo A-I, indicating that clusterin could be incorporated into SMAC without apo A-I. The complement activities of the sera of the patients were higher than those of normal donors. These results may be explained by the deficiencies of apo A-I, apo A-II and HDL in the patients because they were suggested to be the inhibitors of the reactive hemolySis of complement. The elevated complement activities of the patients might be related to the severe atheroselerotic lesions in Tangier disease.
DNA SEQUENCE AND RFLP ANALYSIS FOR THE DEFINITION OF HUMAN COMPLEMENT C4 POLYMORPHISM BASED ON ISOTYPE-SPECIFIC PCR AMPLIFICATION X. Chu, G. Barba, L. Braun-Heimer, C. Rittner, K. Witzel, and P.M. Schneider Institute of Legal Medicine, Johannes Gutenberg-University, Mainz, Germany. The fourth component of the complement system, C4, is encoded by two highly similar, HLA-linked genes, expressing the two different isotypes C4A and C4B. A two-step "nestedPCR" approach was developed to facilitate the isotype-specific amplification of selected exons of the C4 genes. Using PCR primers directed at the sequences coding for residues 11011106 (Chido 4 serological determinant) capable of distinguishing the isotype-specific C4B sequence in the C4d region, in combination with primers from different exon-flanking sequences of the C4 genes, DNA fragments covering exons 10 to 41 from several C4B alleles were amplified. These 5-7 kb long isotype-specific PCR products were then subjected to a second PCR amplification generating fragments of 1-2 kb size suitable for direct DNA sequencing and RFLP analysis. Thus a number of C4B alleles have been sequenced to define the genetic basis of polymorphism. Based on these findings either allele-specific PCR or RFLP analysis of PCR products can be used for the rapid typing of the C4 alleles. [Supported by DFG (Schn 284/3-1 )]
GENERATION OF A FUNCTIONAL DOMAIN OF CRI WITH INCREASED LIGAND BINDING A N D COFACTOR ACTIVITIES. Liliana Clemenza, Richard Hauhart, John Atkinson, Malgorzata Krych. Washington Univ. Sch. of Med., St., Louis, MO 63110, USA There are two distinct binding/active sites in CRI; SITE 1 with short consensus repeats (SCRs) -1/2 and SITE 2 with SCRs-8/9. C4b is a major ligand/substrate for SITE 1. Both C3b and C4b are ligands/substrates for SITE 2. In SITE 1, two conserved amino acids (aa), G in SCR-1 and Y in SCR-2, as well as two nonconserved aa in SCR-2, R and N, were important for C4b binding. Conserved aa are likely a part of a framework of a SCR, whereas nonconserved aa may be involved in direct interactions. We now report that, when these four aa important for C4b binding in SITE 1, were transferred to the homologous positions of SITE 2, functional activity of SITE 2 was increased for both ligands/substrates. As assessed by ELISA over a wide range of ionic strengths, protein carrying this modified SITE 2 bound iC3/C3b and C4b better than the protein with wild type SITE 2. Moreover, it had increased cofactor activity for factor I cleavage of both C3b and C4b. Thus, by genetic manipulation of SCRs, we have constructed a regulator of complement activation (RCA) protein, in this case derived from CRI, with increased functional activity. More effective inhibitors of complement activation can be produced by this strategy.