Polypeptide fragments of the third component of complement in urine of hereditary nephritis patients

Pro-HNP, a urine protein isolated from hereditary nephritis patients, is derived from C3 and resembles the C3c domain. It contains disulfide-linked polypeptides of beta 75, alpha 40, and alpha 28. Plasmin degraded pro-HNP in vitro to HNP, which was also isolated from the urine of patients and which contained disulfide-linked polypeptides of beta 60, alpha 38, and alpha 26, and noncovalently bound polypeptide of beta 17. Amino terminal sequence analyses and amino acid compositions of the seven polypeptides isolated from pro-HNP and HNP show that beta 75 degrades to beta 60 and beta 17 (beta 17 locates at the amino end of beta 75), alpha 40 degrades to alpha 38 (both locate at the carboxyl end of the alpha-chain of C3), and alpha 28 degrades to alpha 26 (both are from the amino end of the alpha'-chain of C3b). These results confirm the enzymatic specificity of plasmin on pro-HNP. In HNP, the half-cystine contents of beta 60, alpha 38, alpha 26, and beta 17 were approximately 3, 12, 3, and 4, respectively. Partial reduction readily released alpha 40 from pro-HNP and alpha 38 from HNP. There were about five intra-chain disulfide bonds in alpha 40 or alpha 38; stepwise reduction of these intra-polypeptide bonds apparently accounted for multiple conformations of alpha 40 or alpha 38.     

Catabolites of the third component of complement in urines of hereditary nephritis patients

Hereditary nephritis protein (HNP), an unusual urine protein from patients with hereditary nephritis (Alport Syndrome), was purified 120-fold to homogeneity. A slightly larger protein, pro-HNP, was similarly purified and was found to be a precursor of HNP. Both pro-HNP and HNP showed immunological identity to the third component of human complement, C3, and to its catabolite C3c. Pro-HNP had a molecular weight of 143,000 and, in equimolar ratio, polypeptide chains or fragments of molecular weights 75,000, 40,000, and 28,000. The largest and smallest chains contained carbohydrate. HNP had a molecular weight of 141,000 and fragments of molecular weights 60,000, 38,000, 26,000, and 17,000 in equimolar ratio; the two smallest fragments contained carbohydrate. Plasmin digestion of pro-HNP showed that the 75,000-Da chain, identical with the intact beta-chain of C3, broke down to the 60,000- and 17,000-Da fragments of HNP. In both pro-HNP and HNP, the polypeptide chains were linked by disulfide bonds, with the exception of the 17,000-Da fragment of HNP. This fragment was readily dissociated from the rest of the HNP molecule in the presence of sodium dodecyl sulfate. Amino acid analyses showed that both pro-HNP and HNP contained approximately 22 half-cystine residues per molecule. Extinction coefficients, epsilon 1% 1cm, at 280 nm were calculated to be 8.5 and 8.8 for pro-HNP and HNP, respectively.     

A mutation causing Alport syndrome with tardive hearing loss is common in the western United States.

Mutations in the COL4A5 gene, located at Xq22, cause Alport syndrome (AS), a nephritis characterized by progressive deterioration of the glomerular basement membrane and usually associated with progressive hearing loss. We have identified a novel mutation, L1649R, present in 9 of 121 independently ascertained families. Affected males shared the same haplotype of eight polymorphic markers tightly linked to COL4A5, indicating common ancestry. Genealogical studies place the birth of this ancestor >200 years ago. The L1649R mutation is a relatively common cause of Alport syndrome in the western United States, in part because of the rapid growth and migratory expansion of mid-nineteenth-century pioneer populations carrying the gene. L1649R affects a highly conserved residue in the NC1 domain, which is involved in key inter- and intramolecular interactions, but results in a relatively mild disease phenotype. Renal failure in an L1649R male typically occurs in the 4th or 5th decade and precedes the onset of significant hearing loss by approximately 10 years.     

The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm.

In Saccharomyces cerevisiae the UPF1 protein is required for nonsense-mediated mRNA decay, the accelerated turnover of mRNAs containing a nonsense mutation. Several lines of evidence suggest that translation plays an important role in the mechanism of nonsense mRNA decay, including a previous report that nonsense mRNAs assemble in polyribosomes. In this study we show that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged protein, UPF1-3EP, retains 86% (+/- 5%) of function. Using immunological detection, we found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus or in the mitochondrion. UPF1-3EP and L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and L1 exhibited similar changes using three different sets of conditions that altered the polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and L1 accumulated in fractions coincident with 80S ribosomal particles. These results suggest that UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal proteins of the 60S and 40S ribosomal subunits, respectively, were on average about 100-fold more abundant than UPF1 mRNA. Assuming that translation rates for L3, S3, and UPF1 mRNA are similar, this result suggests that there are far fewer UPF1 molecules than ribosomes per cell. Constraints imposed by the low UPF1 abundance on the functional relationships between UPF1, polyribosomes, and nonsense mRNA turnover are discussed.     

Single base mutation in alpha 5(IV) collagen chain gene converting a conserved cysteine to serine in Alport syndrome.

We have identified a point mutation in the type IV collagen alpha 5 chain gene (COL4A5) in Alport syndrome. Variant PstI (Barker et al., 1990, Science 248, 1224-1227), and BglII restriction sites with complete linkage with the Alport phenotype have been found in the 3' end of the COL4A5 gene in the large Utah Kindred P. The approximate location of the variant sites was determined by restriction enzyme mapping, after which this region of the gene (1028 bp) was amplified with the polymerase chain reaction (PCR) from DNA of normal and affected individuals for sequencing analysis. The PCR products showed the absence or presence of the variant PstI and BglII sites in DNA from normal and affected individuals, respectively. DNA sequencing revealed a single base change in exon 3 (from the 3' end) in DNA from affected individuals, changing the TGT codon of cysteine to the TCT codon for serine. This single base mutation also generated new restriction sites for PstI and BglII. The mutation involves a cysteine residue that has remained conserved in the carboxyl-end noncollagenous domain (NC domain) of all known type IV collagen alpha chains from Drosophila to man. It is presumably crucial for maintaining the right conformation of the NC domain, which is important for both triple-helix formation and the formation of intermolecular cross-links of type IV collagen molecules.     

Long-term low-dose co-trimoxazole in prophylaxis of childhood urinary tract infection: bacteriological aspects.

The bacteriological consequences of giving long-term low-dose co-trimoxazole to children to prevent reinfection of the urinary tract were studied. Only six "break-through" infections occurred during 2637 child-months of prophylaxis. The children complied well with treatment. During prophylaxis the number of rectal coliform bacilli recovered was greatly and rapidly reduced, but at least 70% of the surviving coliform organisms remained sensitive to the two components of co-trimoxazole. Changes in sensitivity pattern were evident within a month of starting treatment and the proportion of rectal organisms resistant to sulphonamide or trimethoprim did not increase with time. After stopping co-trimoxazole prophylaxis the number of rectal organisms recoverable returned rapidly to normal, as did their sensitivities to trimethoprim and sulphonamide. Further episodes of urinary tract infection developing after prophylaxis was stopped were caused by organisms sensitive to a wide range of antimicrobial agents, including trimethoprim.