sodium-dodecyl-sulfate and Syndrome

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure with significant predisposition to the development of poor prognosis myelodysplasia and leukemia, exocrine pancreatic failure and metaphyseal chondrodysplasia. Although the SBDS gene mutated in this disorder is highly conserved in Archaea and all eukaryotes, the function is unknown. To interpret the molecular consequences of SDS-associated mutations, we have solved the crystal structure of the Archaeoglobus fulgidus SBDS protein orthologue at a resolution of 1.9 angstroms, revealing a three domain architecture. The N-terminal (FYSH) domain is the most frequent target for disease mutations and contains a novel mixed alpha/beta-fold identical to the single domain yeast protein Yhr087wp that is implicated in RNA metabolism. The central domain consists of a three-helical bundle, whereas the C-terminal domain has a ferredoxin-like fold. By genetic complementation analysis of the essential Saccharomyces cerevisiae SBDS orthologue YLR022C, we demonstrate an essential role in vivo for the FYSH domain and the central three-helical bundle. We further show that the common SDS-related K62X truncation is non-functional. Most SDS-related missense mutations that alter surface epitopes do not impair YLR022C function, but mutations affecting residues buried in the hydrophobic core of the FYSH domain severely impair or abrogate complementation. These data are consistent with absence of homozygosity for the common K62X truncation mutation in individuals with SDS, indicating that the SDS disease phenotype is a consequence of expression of hypomorphic SBDS alleles and that complete loss of SBDS function is likely to be lethal.

Topics: Alleles; Amino Acid Sequence; Archaeoglobus fulgidus; Blotting, Western; Cell Cycle; Crystallography, X-Ray; DNA Mutational Analysis; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Epitopes; Escherichia coli; Flow Cytometry; Genetic Complementation Test; Guanidine; Homozygote; Humans; Leukemia; Models, Molecular; Molecular Sequence Data; Mutation; Phenotype; Protein Binding; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Proteins; RNA; RNA, Messenger; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Sodium Dodecyl Sulfate; Syndrome; Time Factors

Patients with the heparin-induced thrombocytopenia syndrome (HIT) have heparin-associated antibodies (HAb+), which, in the presence of heparin, are responsible for platelet activation and aggregation. This study addressed the questions: (1) are the antibodies specific for heparin; and (2) how do the antibodies cause platelet aggregation?. Plasmas from 79 patients with HIT were divided into seven plasma samples: HAb+ plasma sample 1 (24 pooled plasmas); HAb+ plasma sample 2 (50 pooled plasmas); and HAb+ plasma samples 3 through 7 (individual plasmas). Normal patient plasmas were used as controls (HAb-).. All seven HAb+ plasma samples caused platelet aggregation (PLA) in the presence of heparin and formed a precipitation line with heparin in gel immunodiffusion plates (HAb- plasmas did neither). The HAb+ plasma samples reacted with heparin, as determined by immunoprecipitation in sodium dodecylsulfate-polyacrylamide gel, with the production of a band at 50 kd (no band with HAb- plasmas). The plasma samples 1 and 2 were passed over heparin sepharose beads three times; the unabsorbed plasmas produced 3+ PLA, the first effluent produced 2+ PLA, and the second and third effluents produced no PLA. The heparin sepharose beads stained 3+, 2+, and 1+, after the respective passages, with fluorescein-labeled goat sera containing anti-human immunoglobulin G antibody. HAb+ plasma samples were digested with pepsin to separate the F(ab')2 fragments from the Fc fragments. The F(ab')2 fragments reacted with heparin as determined by immunoprecipitation in sodium dodecylsulfate-polyacrylamide gel with the production of a band at 25 kd, but did not cause PLA in the presence of heparin.. Patients with HIT have heparin-specific antibodies that react with heparin in a classic F(ab')2 reaction and require the Fc fragment for platelet aggregation.

Topics: Absorption; Antibodies; Electrophoresis, Polyacrylamide Gel; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Heparin; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Platelet Activation; Platelet Aggregation; Precipitin Tests; Sepharose; Sodium Dodecyl Sulfate; Syndrome; Thrombocytopenia

Carbonic anhydrase II (CA II) deficiency has been shown to be the primary defect in the recessively inherited syndrome of osteopetrosis with renal tubular acidosis. Until now, the absence of CA II in kidney of CA II-deficient patients has not been shown directly, and the status of the membrane-associated CA in kidney of CA II-deficient patients has been unclear. To address these questions, we analyzed urinary membranes and soluble fractions from normal and CA II-deficient subjects. The CA activity in membrane fractions of normal urine was found to comprise two components--(i) a vesicle-enclosed, sodium dodecyl sulfate (SDS)-sensitive fraction, which was shown immunochemically to be the 29-kDa CA II, and (ii) an SDS-resistant fraction, which was due to native and cleaved forms of the 35-kDa, membrane-anchored isozyme CA IV. Urinary membranes from CA II-deficient patients showed little or no SDS-sensitive activity and no immunoreactivity for CA II, providing direct evidence that their mutation, which produces CA II deficiency in erythrocytes, also affects CA II in kidney. CA IV activity and immunoreactivity were present in normal amounts in urinary membranes from CA II-deficient patients. We conclude from the enzymatic and immunological evidence presented that both CA II and CA IV are present in urinary membranes from normal subjects, that renal CA IV is present but renal CA II is absent in urinary membranes from patients with the CA II-deficiency syndrome, and that the methods presented should be useful in studying renal CA II and renal CA IV in other disorders of impaired bicarbonate reabsorption.

Topics: Acidosis, Renal Tubular; Adolescent; Adult; Carbonic Anhydrases; Child; Electrophoresis, Polyacrylamide Gel; Female; Humans; Isoenzymes; Kinetics; Male; Microvilli; Middle Aged; Osteopetrosis; Reference Values; Sodium Dodecyl Sulfate; Syndrome; Urine

Using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, a high molecular weight Ricinus communis I (RCA I)-binding glycoprotein (approx. Mr 370000) has been identified in human muscle that is consistently altered or absent in muscle from patients with Duchenne muscular dystrophy (DMD). In addition, a Mr 54000 RCA I-binding glycoprotein was identified in 4 out and 8 DMD muscle samples that was not present in normal muscle. The possibility that the Mr 370000 glycoprotein could be a muscle membrane glycoprotein which is altered or absent in DMD is discussed.

Topics: Adolescent; Adult; Child; Child, Preschool; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Infant; Membrane Proteins; Molecular Weight; Muscles; Muscular Dystrophies; Sodium Dodecyl Sulfate; Syndrome

The glycoprotein profile of Bernard-Soulier platelets was examined by labeling washed platelets with periodate 3H-sodium borohydride, a procedure that labels greater than 30 glycoproteins on the membrane surface of normal platelets. Three Bernard-Soulier patients were studied; two were siblings and the third was unrelated. The platelet protein and glycoprotein profiles were evaluated under nonreduced and reduced conditions using 5%-15% exponential SDS-polyacrylamide gel electrophoresis. The two siblings completely lacked glycoprotein Ib (GPIb). The unrelated patient had congruent to 7% of the normal level. This was confirmed by two-dimensional nonreduced-reduced SDS-polyacrylamide gel electrophoresis, a procedure that allows clear separation of the disulfide-linked subunits of GPIb, GPIb alpha (mol wt 145,000), and GPIb beta (mol wt 25,000) from other membrane glycoproteins. On one-dimensional analysis, Bernard-Soulier's syndrome (BSS) platelets also lacked the peripheral membrane glycoprotein, GPV (mol wt 82,000) and a low molecular weight glycoprotein, GPIX, (nonreduced or reduced, mol wt congruent to 22,000). The two-dimensional gel system also revealed the absence of a minor glycoprotein with a molecular weight of congruent to 100,000 (GP 100). Quantitation of these proteins solubilized from electrophoretograms showed that the siblings' parents had congruent to 50% levels of GPIb, GPIX, and GP 100. A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets. The combined results suggest that the apparent genetic absence of multiple proteins in Bernard-Soulier platelets is due, in part, to the presence in normal platelets of a tight membrane complex between glycoprotein Ib and at least one of the other absent glycoproteins.

Topics: Adolescent; Adult; Blood Platelet Disorders; Blood Platelets; Electrophoresis, Polyacrylamide Gel; Female; Glycoproteins; Humans; Male; Membrane Proteins; Periodic Acid; Platelet Membrane Glycoproteins; Sodium Dodecyl Sulfate; Syndrome

Spectrophotometric and gas-liquid chromatographic analyses on the carbohydrate moiety of tryptic erythrocyte glycopeptides from persons with Tn-syndrome reveal a selective lowering of the galactose and sialic acid content, the degree being dependent on the percentage of polyagglutinable cells. Alkaline borohydride specifically releases N-acetylgalactosaminitol, and the amount is correlated to the percentage of pathological acetylgalactosaminitol, and the amount is correlated to the percentage of pathological erythrocytes. It is concluded that the alkali-labile carbohydrate chains of Tn-polyagglutinable red cells solely consist of N-acetylgalactosamine linked to serine or threonine. Experiments with heterophile agglutinins whose specificity is known are in line with the above-mentioned results. As judged from SDS-polyacrylamide gel electrophoresis the three major membrane glycoproteins are affected to a different extent by the defect.

Topics: Anemia, Hemolytic; Antibodies, Heterophile; Blood Glucose; Blood Protein Electrophoresis; Cell Membrane; Chromatography, Gas; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Glycopeptides; Hemagglutination; Hemagglutination Tests; Hexoses; Humans; Leukopenia; Molecular Weight; Sialic Acids; Sodium Dodecyl Sulfate; Syndrome; Thrombocytopenia; Trypsin

Topics: Blood Platelets; Centrifugation; Electrophoresis, Polyacrylamide Gel; Female; Glycoproteins; Humans; Male; Membranes; Molecular Weight; Purpura, Thrombocytopenic; Sialic Acids; Sodium Dodecyl Sulfate; Syndrome; Temperature; Trypsin; Ultrasonics