Polarity of human replication protein A binding to DNA

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3′-end of the upstream oligonucleotide or at the 5′-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5′-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3′-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer–template systems containing 5′- or 3′-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.     

Salivary IgA subclasses and infection risk in elite swimmers.

The concentrations of total IgA, IgA1 and IgA2 were measured in saliva collected from 25 elite swimmers in the early and late phases of a 7 month training season and compared with the number of respiratory infections during the season. The IgA1 concentrations in the early phase of the training season were significantly associated (P = 0.01) with the number of respiratory infection episodes during the training season. The lower the concentration of IgA1, the greater the number of infection episodes. Swimmers with four or more infections during the training season had significantly lower salivary IgA1 concentrations than those with less than four infection episodes (P = 0.01). The proportion of IgA1 in the saliva of the elite swimmers (80%) was higher than for normal non-exercising adults (60%). A small proportion of athletes had salivary IgA2 concentrations below the detection limit of the assay and the mean concentration of IgA2 was significantly lower than the concentrations for a normal adult population (P = 0.01). This study suggests that measurement of IgA subclasses, in particular IgA1, at the commencement of a training season may predict infection risk in elite swimmers.     

Agaricus bisporus lectin binds mainly O-glycans but also N-glycans of human IgA subclasses

The primary interaction between purified Agaricus bisporus lectin (ABL) and human IgA subclasses was studied by ABL-affinity chromatography, dot blot assay and competitive enzyme-lectin assay, considering that ABL could be an alternative tool for detection of IgA1 O-glycans. Both secretory IgA subclasses bound to ABL-Sepharose and the IgA2 subclass (which contains only N-glycans) was recovered with a high degree of purity when NH4OH was used as eluent. ABL-Ig interaction was also observed by dot blot assays using ABL-peroxidase against monoclonal IgA1 k Pan, IgA2m(1)k Gir, IgA2m(2)k Bel, secretory IgA2 and normal IgG (also contains only N-glycans). When these immunoglobulins were enzymatically treated with peptide N-glycosidase F (N-glycan hydrolysis), the ABL-IgA2 and -IgG interaction did not occur while IgA1 maintained a high degree of interaction with ABL. Also, the ABL-IgA interaction was observed by competitive enzyme-lectin assay, and when IgA1 subclass was treated with endo-α-N-acetylgalactosaminidase for O-glycans hydrolysis, the reactivity with ABL was very low. We conclude that the complementary use of ABL and peptide N-glycosidase F could be a useful tool to assess the O-glycosylation state of human IgA1 subclass, which is of relevant importance in the effector functions of immunoglobulins. Abbreviations: ABL, Agaricus bisporus lectin; α1 and α2, heavy chains from human IgA1 and IgA2; C1-3α, constant domains (1–3) of heavy chains from human IgA; ECL, Erythrina cristagalli lectin; EEO, electroendoosmosis; EIA, enzyme immunoassay; ELA, enzyme lectin assay; Ig, immunoglobulin; HRP, horseradish peroxidase; ID50, 50% inhibitory dose; Ka, affinity constant; O.D., optical density; PBS, phosphate buffered saline; PBS-t, phosphate buffered saline with Tween 20; PNGase F, peptide N-glycosidase F; RID, radial immunodiffusion; SD, standard deviation; TBS, Tris-HCL buffered saline; TBS-t, Tris-HCI buffered saline with Tween 20; T-disaccharide, Thomsen-Friedenreich disaccharide This revised version was published online in November 2006 with corrections to the Cover Date.     

Quantitative analysis of IgA1 binding protein prepared from human serum by hypoglycosylated IgA1/Sepharose affinity chromatography

The binding protein to a hypoglycosylated IgA1/Sepharose (IgA1-BP) could be prepared from human sera. IgG was a major component in the IgA1-BP. A Protein A column was used to remove the IgG; however, about half of the IgA1-BP was passed from the column [Biochem. Biophys. Res. Commun., 264 (1999) 424]. Quantitative analysis of the passed fraction (PAP) by laser nepherometry indicated that it was composed of a fairly large amount of IgA, IgM and complement C3 besides IgG. The relative content of IgG:IgA:IgM:C3:C4 was 25:10:41:22:2 in the PAP fraction. Meanwhile, the Protein A bound-fraction was essentially composed of IgG (78%) and IgM (19%). The total amount of IgA1-BP was not different between the sera from IgA nephropathy patients and other nephropathy patients. With respect to the IgA content in the IgA1-BP from IgA nephropathy patients, it was significantly higher than that from other nephropathy patients. It was found that the IgA1-BP from some IgA nephropathy patients contained a few micrograms of aberrant IgA per ml of serum. Thus, the obtained results suggested the preferential deposition of the self-aggregated IgA composed of hypoglycosylated IgA1 and co-deposition of IgG, IgM and C3 in the glomeruli in an IgA nephropathy patient.     

The binding of penicillin antibiotics to a human liver protein.

A protein has been isolated from postmortem human livers which binds the penicillin analogs, dicloxacillin, nafcillin and benzylpenicillin, as well as oleic acid. Its molecular weight is in the range 17,000–20,000 and its pI is 5.9. It appears to be similar to a fatty acid-binding protein previously isolated from rat tissues.     

Protein Rou. A human IgA hybrid

Protein Rou is a human IgA2 myeloma protein that carries the isoallotype marker n A2m(2). Partial amino acid sequence of its H chain (alpha) shows that the hinge region and the CH2 domain are homologous to alpha 2-chain and the CH1 and the CH3 domains homologous to alpha 1. Moreover, the CH1 domain contains the H-L disulfide bond identical to alpha 1. It is concluded that Rou H chain is a hybrid molecule caused by a recombination between alpha 1 and alpha 2 genes. The recombination event occurred between alpha 1-exon 1 and alpha 2-exon hinge and corresponds to position 222-223 of the alpha-chain.     

FiberID--a technique to identify fibrous protein subclasses

Fibrous proteins such as collagen, silk, and elastin play critical biological roles, yet they have been the subject of few projects that use computational techniques to predict either their class or their structure. In this article, we present FiberID, a simple yet effective method for identifying and distinguishing three fibrous protein subclasses from their primary sequences. Using a combination of amino acid composition and fast Fourier measurements, FiberID can classify fibrous proteins belonging to these subclasses with high accuracy by using two standard machine learning techniques (decision trees and Naïve Bayesian classifiers). After presenting our results, we present several fibrous sequences that are regularly misclassified by FiberID as sequences of potential interest for further study. Finally, we analyze the decision trees developed by FiberID for potential insights regarding the structure of these proteins. Proteins 2007. © 2006 Wiley‐Liss, Inc.     

Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B

Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1-3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections.     

Heterogeneity in the kinetics of oxygen binding to partially reduced human methemoglobin. A pulse-radiolysis study of oxygenated solutions of methemoglobin

The pulse-radiolysis technique has been introduced because it permits a rapid reduction (in a few microseconds) of one heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the binding of oxygen to this particular valence intermediate (Hb3+) with one reduced alpha or beta subunit has been studied. It appears that the hydrated electrons preferentially reduce one type of subunit of methemoglobin at acid and neutral pH-values as is shown by the biphasic behaviour of Hb3+ on oxygenation. The second-order on-rate constants measured for the binding of oxygen to Hb3+ are 14 +/- 3 mM-1 ms-1 and 56 +/- 9 mM-1 ms-1, respectively. The relative contribution of the faster fraction is about 0.63 +/- 0.08 of the total oxygenation process. A comparison of the kinetic absorbance difference spectrum for the reduction of methemoglobin with the static difference spectrum of deoxyhemoglobin and methemoglobin in the Soret-region revealed a decreased absorbance of the unliganded subunit of Hb3+ at 430 nm. This fact suggests that Hb3+ is in the relaxed quaternary conformation, which is in agreement with the observed on-rate constants.     

Stereoselective binding of disopyramide to human plasma protein

The binding of racemic disopyramide and its two enantiomers to protein were compared in two samples of human plasma, two samples of freshly drawn serum and in a solution of alpha 1-acid glycoprotein. The binding of S(+)-disopyramide was higher at all concentrations as compared to to R(?)-disopyramide, and the binding of the racemate was intermediate. Differences in binding were due to differences in the association constant.     

The modification of human immunoglobulin binding to staphylococcal protein A using diethylpyrocarbonate

Human IgG subclasses 1, 2, and 4, as well as proteins of the IgG3 subclass that are allotype G3m (s+t+), bind avidly to staphylococcal protein A by means of their Fc portion. Proteins of the IgG3 subclass that are allotype G3m (s-t-) do not bind. The importance of a histidine residue at position 435 has been implicated from comparison of amino acid sequences of immunoglobulins that bind with those that do not bind to staphylococcal protein A, as well as from crystallographic data. Modification of histidines at a low concentration of diethylpyrocarbonate successfully and reversibly alters the binding of immunoglobulins to staphylococcal protein A with only minimal change in the antigenic properties. This method provides strong evidence for the critical importance of histidine in the binding of immunoglobulins to staphylococcal protein A.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Cytosolic protein binding to band-3 protein inhibits endocytosis of isolated human erythrocyte membranes.

Recent studies of haemoglobin binding to the cytoplasmic side of the erythrocyte membrane have shown that the predominant high-affinity interaction occurs with the major integral membrane protein known as band-3 protein and that this interaction may occur within the intact erythrocyte in a manner regulated by cell pH. We report here that haemoglobin and glyceraldehyde 3-phosphate dehydrogenase binding to band-3 protein in isolated membranes can inhibit endocytosis during vesiculation in vitro. The specificity of this effect was demonstrated by showing that myoglobin, which has an affinity for the membrane fully one to two orders of magnitude lower than that for haemoglobin, does not inhibit endocytosis.     

A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10¹⁰M^–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.     

Covalent binding of human thrombin to a human endothelial cell-associated protein

Binding of 125I-thrombin to endothelial cells derived from human umbilical vein was studied in tissue culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed covalent binding of thrombin in a 72-kDa complex. This binding is specific and requires the catalytically active site of the enzyme. Formation of the complex could be detected as early as 3 min after addition of thrombin or with a thrombin concentration as low as 0.5 nM. This irreversible binding exhibits thrombin dose-dependence and reaches maximum levels at a concentration of 50 nM (10 fmol/10(5) cells). Some characteristics of the 72-kDa complex were compared to those of the complexes formed between thrombin and protease nexin originating from fibroblasts or platelets: (i) its electrophoretic mobility on SDS-PAGE is identical to that of the thrombin-platelet protease nexin complex, (ii) heparin prevents the appearance of the complex on the cell surface, (iii) plasmin in a 100-fold molar excess prevents the covalent linkage of thrombin, suggesting that the protease specificity of the endothelial component involved in the complex might not be restricted to thrombin. Yet no release, nor any secretion of the endothelial protein, could be detected. These results indicate that active thrombin binds covalently to a specific endothelial protein that is in several respects similar to fibroblast or platelet protease nexin and provides a thrombin binding site distinct from thrombomodulin and glycosaminoglycans.