d-Amino acid oxidase deficiency is caused by a large deletion in the Dao gene in LEA rats

d-Amino acids, enantiomers of l-amino acids, are increasingly recognized as physiologically active molecules as well as potential biomarkers for diseases. d-Amino acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids and is present in a wide variety of organisms from yeasts to humans. Previous studies indicated that LEA rats lacked DAO activity, and levels of d-Ser and d-Ala were markedly increased in their tissues, suggesting a mutated locus responsible for the lack of Dao activity (ldao) existed in the LEA genome. Sequence analysis identified deletion breakpoints located in intron 4–5 of the Dao gene and intron 1–2 of the Svop gene, resulting in a 54.1-kb deletion which encompassed exons 5–12 of the Dao gene and exons 2–16 of the Svop gene. We developed a novel congenic rat strain, F344-Dao^ldao, harboring the Dao^ldao mutation from LEA rats delivered onto the F344 genetic background. Compared to the parental F344 strain, in F344-Dao^ldao rats d-Ala was markedly increased in both cerebrum and cerebellum, while d-Ser content was increased in cerebellum but not cerebrum. d-Ala, d-Ser, d-Pro and d-Leu levels were also elevated in F344-Dao^ldao plasma. F344-Dao^ldao rats represent a novel model system that will aid in elucidating the physiological functions of d-amino acids in vivo. (203 words).     

Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.     

Inhibition of bFGF activity by complement C1s: covalent binding of C1s with bFGF

The first complement component C1s formed large aggregates with bFGF when bFGF and C1s were incubated at 37°C overnight. Under non-reducing conditions, a part of the aggregates did not penetrate into 5% polyacrylamide gel in the presence of SDS, and the rest penetrated into 5% gel but not into 12% gel. The aggregates were dissociated into monomers by reducing with 2-mercaptoethanol. Both active and inactive C1s formed aggregates with bFGF. In addition, a portion of bFGF was degraded by active C1s but not by inactive C1s. Aggregates were not formed when 2-mercaptoethanol (2 mM &base;) was added to the incubation mixture. After the incubation with C1s the growth-stimulating activity of bFGF was measured by using human umbilical vein endothelial cells (HUVEC) as indicator cells. The aggregate formation between C1s and bFGF significantly reduced the activity of bFGF. © 1998 John Wiley & Sons, Ltd.     

Kinetics of interaction of C1 inhibitor with complement C1s

The kinetics of inhibition of the complement serine protease, C1s, by its only known inhibitor, C1 inhibitor, have been measured by a variety of methods. One method continuously monitors the loss of esterolytic activity with a synthetic substrate coupled to a chromogen while another monitors the formation of a stable (covalent) complex by high-pressure size-exclusion chromatography under dissociating conditions. Additional methods employ fluorescence probes to follow the formation of bimolecular complexes but are not expected to distinguish between covalent product and noncovalent (reversible) intermediates. There was good agreement between rate constants obtained by the various methods over a broad range of inhibitor concentrations, suggesting that noncovalent intermediates do not accumulate to a significant extent. The reaction appears to be pure second order with a bimolecular rate constant of 6.0 X 10(4) M-1 s-1 at 30 degrees C, independent of Ca2+, and an activation energy of 11.0 kcal/mol. The rate increases up to 35-fold in the presence of heparin which was shown to bind to all three components (enzyme, inhibitor, and complex) with similar affinity (Kd = 2.0-3.3 microM). The fluorescent probe 1,1'-bis(anilino)-4-,4'-bi(naphthalene)-8,8'-disulfonate [bis(ANS)] bound to the complex with Kd = 0.26 microM under conditions where the individual components had little affinity for the dye, consistent with the generation of one or more hydrophobic binding sites on the protein surface during complex formation.     

Nonsense mutation in exon 4 of human complement C9 gene is the major cause of Japanese complement C9 deficiency

Deficiency of the ninth component of human complement (C9) is the most common complement deficiency in Japan but is rare in other countries. We studied the molecular basis of C9 deficiency in four Japanese C9-deficient patients who had suffered from meningococcal meningitis. Direct sequencing of amplified C9 cDNA and DNA revealed a nonsense substitution (CGA→TGA) at codon 95 in exon 4 in the four C9-deficient individuals. An allele-specific polymerase chain reaction system designed to detect exclusively only one of the normal and mutant alleles indicated that all the four patients were homozygous for the mutation in exon 4 and that the parents of patient 2 were heterozygous. The common mutation at codon 95 in exon 4 might be responsible for most Japanese C9 deficiency. Received: 28 December 1997 / Accepted: 25 February 1998     

Restoration of complex V deficiency caused by a novel deletion in the human TMEM70 gene normalizes mitochondrial morphology

We report a fragmented mitochondrial network and swollen and irregularly shaped mitochondria with partial to complete loss of the cristae in fibroblasts of a patient with a novel TMEM70 gene deletion, which could be completely restored by complementation of the TMEM70 genetic defect. Comparative genomics analysis predicted the topology of TMEM70 in the inner mitochondrial membrane, which could be confirmed by immunogold labeling experiments, and showed that the TMEM70 gene is not restricted to higher multi-cellular eukaryotes. This study demonstrates that the role of complex V in mitochondrial cristae morphology applies to human mitochondrial disease pathology.     

Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1s, C1r2-C1s2 and C1

Photosynthetic membranes contain considerable regions of high surface curvature, notably at their margins, where the average radius of curvature is about 10 nm. The proportion of total membrane lipid in the outer and inner thylakoid margin monolayers is estimated at 21% and 13%, respectively. The major thylakoid lipid, monogalactosyldiacylglycerol, is roughly cone-shaped and will not form complete lamellar bilayer phases, even in combination with other thylakoid lipids. It is proposed that this galactolipid plays a role in: (a) stabilising regions of concave curvature in thylakoids; and (b) packaging hydrophobic proteins in planar bilayer regions by means of inverted micelles. This model predicts substantial asymmetries in the distribution of lipids both across and along the thylakoid bilayer plane.     

Calcium-linked self-association of human complement C1s.

The weight-average molecular weight of C1s, an activated serine protease subcomponent of human complement C1, has been measured by means of sedimentation equilibrium over a wide range of both protein and calcium ion concentrations. The combined data may be accounted for quantitatively by a simple model for Ca(2+)-dependent self-association of C1s to a dimer. According to this model, the monomer contains a single Ca2+ binding site with K approximately equal to 3 x 10(5) M-1, and the dimer contains three independent Ca binding sites, two having a Ca2+ affinity lower than that of the monomer (K approximately equal to 3 x 10(4) M-1). The third binding site in the dimer, which presumably lies at the interface between the two amino-terminal alpha domains, has a higher Ca2+ affinity (K approximately equal to 1 x 10(8) M-1) and provides the driving force for C1s dimerization in the presence of calcium.     

Identification of the disulfide bonds of human complement C1s

C1s, one of the three subcomponents of C1, the first component of the complement system, is a complex serine protease. To determine the disulfide-bonding pattern, fragments of C1s were generated by cleavage with pepsin, thermolysin, or subtilisin. Disulfide bonds have been identified by several methods, for example, direct observation of the phenylthiohydantoin derivative of cystine during Edman degradation of isolated peptides and placement in the known cDNA sequence. All of the 26 half-cystines are linked in disulfide bonds occurring at positions 50-68, 120-132, 128-141, 143-156, 160-187, 219-236, 279-326, 306-339, 344-388, 371-406, 410-534, 580-603, and 613-644. All of the disulfide bonds of the earlier described substructures of C1s, the EGF-homologous part, the two SCR units, and the two domains typical for C1s and C1r are localized within these domains.     

New possibilities of treating acute angioedema caused by C1-inhibitor deficiency

The authors discuss diagnostic difficulties in 12 cases of hereditary angioneurotic edema due to C1-esterase inhibitor (C1-INH deficiency). Emphasis is on the treatment of the acute attacks with intravenous infusions of C1-inhibitor concentrate (Boehring, West Germany). This proved to be a very efficient and safe therapy, leading to a prompt disappearance of all clinical symptoms. Throughout 12 months following the infusions, indices of the liver function remained within the normal range, and anti-Hbs and anti-HIV tests were negative.     

Characteristics of complement subcomponents C1r and C1s synthesized by Hep G2 cells.

The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2.     

An unusual combined insertion/deletion polymorphism in intron 10 of the human complement C6 gene

Investigation of intron 10 of the human complement C6 gene revealed an unusual combined insertion/deletion polymorphism at position 493: the subsequent 6 bp is deleted and is substituted by a different 26 bp, giving a net gain of 20 bp. The variant shows autosomal co-dominant inheritance. The 26 bp insertion is homologous to a human endogenous retrovirus-type sequence and could tentatively be ascribed to a retroposon. Alternatively, the presence of three copies of a 5 bp direct repeat, an 8 bp palindrome and a 12 bp split symmetrical element could suggest an endogenous, sequence-mediated mutational process. Polymorphisms of this type are extremely rare, although there are several examples of such mutations causing disease. Received: 30 September 1996 / Accepted: 12 February 1997     

Functional role of the linker between the complement control protein modules of complement protease C1s

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of C (C1). Its catalytic domain comprises two complement control protein (CCP) modules connected by a four-residue linker Gln340-Pro-Val-Asp343 and a serine protease domain. To assess the functional role of the linker, a series of mutations were performed at positions 340-343 of human C1s, and the resulting mutants were produced using a baculovirus-mediated expression system and characterized functionally. All mutants were secreted in a proenzyme form and had a mass of 77,203-77,716 Da comparable to that of wild-type C1s, except Q340E, which had a mass of 82,008 Da, due to overglycosylation at Asn391. None of the mutations significantly altered C1s ability to assemble with C1r and C1q within C1. Whereas the other mutations had no effect on C1s activation, the Q340E mutant was totally resistant to C1r-mediated activation, both in the fluid phase and within the C1 complex. Once activated, all mutants cleaved C2 with an efficiency comparable to that of wild-type C1s. In contrast, most of the mutations resulted in a decreased C4-cleaving activity, with particularly pronounced inhibitory effects for point mutants Q340K, P341I, V342K, and D343N. Comparable effects were observed when the C4-cleaving activity of the mutants was measured inside C1. Thus, flexibility of the C1s CCP1-CCP2 linker plays no significant role in C1 assembly or C1s activation by C1r inside C1 but plays a critical role in C4 cleavage by adjusting positioning of this substrate for optimal cleavage by the C1s active site.     

Conformational changes of the subunits C1q, C1r and C1s of human complement component C1 demonstrated by 125I labeling

C1s and C1r proenzymes and enzymes (C1s, C1r) and C1q were labeled with 125I. The distribution of the 125I label between H- and L-chain of C1s was only slightly dependent on the state of activation of C1s, and approx. 90% of the label was found in the H-chain. In the C1r proenzyme molecules 50% of the label was incorporated into the H-chain. The C1r H-chain label was reduced to 10% on activation of C1r to C1r, while the L-chain label increased to 90% of the total label. The presence of either C1s, C1q or C1qs during labeling reduced the C1r H-chain level, although C1r remained in the proenzyme form. The presence of C1s or C1rs enhanced the 125I uptake of C1q in Ca2+ or EDTA medium. This was unexpected because one would have anticipated a diminution of the C1q label due to the apposition of C1r and C1s, similarly as it occurs during C1rs complex and C1s dimer formation for the H-chain label of C1s. The results show that C1r and C1q alter their conformation during activation and C1 complex formation.     

Selective expression of hepatocellular membrane proteins in mice homozygous for a lethal chromosomal deletion

Previous studies of mice homozygous for one of several overlapping radiation-induced deletions in chromosome 7 revealed reduced expression of a number of hepatocyte proteins. These proteins include the plasma membrane receptors for insulin, epidermal growth factor, and glucagon, all of which are reduced in number by over 70% with no change in apparent affinity constants. It is not known whether all or only select liver cell surface proteins are so affected in newborn deletion homozygotes. In the present study, we investigated expression of two additional, functionally distinct intrinsic hepatocellular membrane binding proteins: the hepatic binding protein (HBP: the receptor for asialoglycoproteins), and the organic anion binding protein (OABP) which may play a role in organic anion transport. Immunoblot analysis showed the content of OABP and HBP in neonatal mutants to be identical to that in controls. As compared to adults, neonates showed levels of only about 8% of HBP and 75% of OABP binding proteins. Assays of HBP binding activities confirmed these results. The normal levels of these hepatocyte binding proteins in the deletion homozygotes suggest that the DNA sequences deleted in these mutants do not regulate all genes encoding such proteins but only a selected number of them.