Biomedical subjects

R J Simpson

Publications and source records attributed to R J Simpson.

At least 73 records · Page 4Linked to original sources

PubMed1998

Physicochemical characterization of an antagonistic human interleukin-6 dimer.

A noncovalently bound dimeric form of recombinant human IL-6 interleukin-6 (IL-6D) was shown to be an antagonist for IL-6 activity, in a STAT3 tyrosine phosphorylation assay using HepG2 cells, under conditions where it does not dissociate into monomeric IL-6 (IL-6M). The fluorescence from Trp157, the single tryptophan residue in the primary sequence of IL-6, is altered in IL-6D, where the wavelength maximum is blue-shifted by 3 nm and the emission intensity is reduced by 30%. These data suggest that Trp157 is close to, but not buried by, the dimer interface. Both IL-6D and IL-6M are compact molecules, as determined by sedimentation velocity analysis, and contain essentially identical levels of secondary and tertiary structure, as determined by far- and near-UV CD, respectively. IL-6D and IL-6M show the same susceptibility to limited proteolytic attack, and exhibit identical far-UV CD-monitored urea-denaturation profiles with the midpoint of denaturation occurring at 6.0 +/- 0.1 M urea. However, IL-6D was found to dissociate prior to the complete unfolding of the protein, with a midpoint of dissociation of 3 M urea, suggesting that dissociation and dimerization occur when the protein is in a partially unfolded state. Based on these results, we suggest that IL-6D is a metastable domain-swapped dimer, comprising two monomeric units where identical helices from each protein chain are swapped through the loop regions at the "top" of the protein (i.e., the region of the protein most distal from the N- and C-termini). Such an arrangement would account for the antagonistic activity of IL-6D. In this model, receptor binding site I, which comprises residues in the A/B loop and the C-terminus of the protein, is free to bind the IL-6 receptor. However, site III, which includes Trp157 and residues in the C/D loop and N-terminal end of helix D, and perhaps site II, which comprises residues in the A and C helices, are no longer able to bind the signal transducing component of the IL-6 receptor complex, gp130.

Carcinoma, Hepatocellular↗

PubMed1998

Inhibition of growth and aggregation of calcium oxalate crystals in vitro--a comparison of four human proteins.

The aim of this study was to compare directly, in the absence of interfering contaminants, the inhibitory effects of Tamm-Horsfall glycoprotein (THG), human serum albumin (HSA), alpha1-microglobulin and prothrombin fragment 1 (PTF1) on calcium oxalate crystallization. These proteins have been detected in urinary calculi, and with the exception of THG in calcium oxalate crystals generated from undiluted human urine. THG was isolated from the urine of healthy men, while PTF1 was purified from Prothrombinex-HT, a human blood concentrate; HSA and alpha1-microglobulin were obtained from commercial sources. The effects of these proteins were determined, separately, at the same final concentration (32 nM) on calcium oxalate crystallization in a seeded, inorganic reaction system, using Coulter Counter and [14C]oxalate analysis. Analysis of [14C]oxalate data showed that THG, HSA and alpha1-microglobulin had no measurable effect on deposition of calcium oxalate. However, PTF1 significantly inhibited mineral deposition by 19.6%. The average size of the particles precipitated was reduced from the control value of 8.6 microm to 7.3, 5.9, 5.6 and 4.0 microm in the presence of alpha1-microglobulin, HSA, THG and PTF1 respectively. These findings were confirmed by scanning electron microscopy, which also revealed that the smaller particles deposited in the presence of the proteins resulted from reduced crystal aggregation rather than a decrease in the size of the individual crystals. It was concluded that, on a molar basis, PTF1 is a more potent inhibitor of calcium oxalate crystal aggregation than THG, HSA and alpha1-microglobulin. Moreover, unlike those proteins it significantly inhibits the deposition of calcium oxalate. These findings have implications for the putative role of urinary proteins in the formation of calcium oxalate stones.

Alpha-Globulins↗

PubMed1998

Identification and characterization of two distinct truncated forms of gp130 and a soluble form of leukemia inhibitory factor receptor alpha-chain in normal human urine and plasma.

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high affinity, functional receptor complex. In this report, we present evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a molecular weight of approximately 100, 000, which is similar to a previously described form of soluble gp130 in human serum, and a previously undescribed small form with a molecular weight of approximately 50,000. Using a panel of monoclonal antibodies raised against the extracellular domain of human gp130, we were able to show that the small form of the urinary gp130 probably contained only the hemopoietin domain. Both forms of gp130 bound LIF specifically and were capable of forming heterotrimeric complexes with soluble human LIF receptor alpha-chain in the presence of human LIF. In addition to the soluble forms of gp130, a soluble form of LIF receptor alpha-chain was also detected in human urine and plasma.

Amino Acid Sequence↗

PubMed1998

Micro-sequencing strategies for the human A33 antigen, a novel surface glycoprotein of human gastrointestinal epithelium.

Monoclonal antibody (mAb) A33, which recognizes a M(r) approximately 43,000 differentiation antigen (A33) expressed in normal human colonic and small bowel epithelium as well as in 95% of colon cancers, shows specific targeting of colon cancer in humans and is currently being evaluated for clinical use. Here, we describe strategies for the purification and structural analysis of the A33 antigen from the human colorectal carcinoma cell lines LIM1215 and SW1222. Edman degradation of the intact protein and nine peptides, derived by proteolytic digestion of the A33 antigen with Asp-N endoproteinase, thermolysin, trypsin and pepsin followed by micropreparative reversed-phase high-performance liquid chromatography, allowed the unambiguous sequence assignment of 153 amino acid residues; these data reveal one N-glycosylation sequeon in Asp-N endoproteinase peptide D4, and a disulfide linkage between peptides D1 and D4. This amino acid sequence information has facilitated the cloning and subsequent sequencing of a cDNA for the A33 antigen which demonstrates that it is a novel human cell surface molecule of the immunoglobulin superfamily.

Amino Acid Sequence↗

PubMed1998

Two-dimensional electrophoretic analysis of mixed lineage kinase 2 N-terminal domain binding proteins.

The mixed lineage kinase 2 (MLK2) protein contains several structurally distinct domains including an src homology (SH) 3 domain, a kinase catalytic domain, two leucine zippers, a basic motif and a cdc42/rac interactive binding motif. These domains have been recognized mainly for their involvement in protein-protein interactions in signal transduction networks. The SH3 domain in particular has been implicated in control of signaling events. To identify proteins that interact with MLK2, the N-terminal 100 amino acids, including the SH3 domain, were expressed as a glutathione S-transferase (GST) fusion protein. This fusion protein (MLK2N) was used as an affinity ligand to isolate binding proteins from lysates of 35S-radiolabeled MDA-MB231 breast carcinoma cells. When the radiolabeled binding proteins were subjected to 2-DE, proteins of Mr 55,000, 31,500 and 34,000 bound consistently to the MLK2N domain fusion protein, but not to the GST control. Two of the binding proteins were isolated from whole cell lysates by preparative 2-DE and subjected to in-gel digestion and capillary or microbore reverse-phase high performance liquid chromatography (RP-HPLC). Resultant peptides were analyzed by peptide mass fingerprinting, N-terminal Edman degradation or tandem mass spectrometry. The 55,000 protein was identified as the cytoskeletal protein, beta-tubulin, and this was verified by immunoblotting of proteins in the MLK2N binding fraction with anti-tubulin antibodies. The 31,500 protein has been identified as prohibitin, a protein that has been implicated in both signal transduction and cell cycle arrest.

Amino Acid Sequence↗

PubMed1998

Two-dimensional gel database of human breast carcinoma cell expressed proteins: an update.

Previously, we reported a two-dimensional gel map and database with molecular weight/isoelectric point (Mr/pI) loci for 22 proteins expressed in the breast carcinoma cell line, MDA-MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588-598). Here we update this database with Mr/pI loci for a further nine cytoplasmic proteins and three Triton X-114 solubilised membrane proteins from MDA-MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X-114 soluble protein and assigned an Mr/pI locus. During the course of isolating proteins from the Triton X-114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier ampholytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS-polyacrylamide gels after the use of carrier ampholytes for the first dimension. We also report our mass spectrometric-based procedure for identifying two-dimensional electrophoresis (2-DE) gel-resolved proteins, combining in-gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping and electrospray ionisation--ion trap--mass spectrometry.

Amino Acid Sequence↗

PubMed1998

Development of a two-dimensional gel electrophoresis database of human lysosomal proteins.

Two-dimensional gel electrophoresis databases have been generated for a range of tissue cell and fluid types, from a number of species. A major difficulty in the development of such databases is the large number of proteins present in even a single cell type (> 10,000) and the low levels of many of these proteins. One approach to overcome these difficulties is to fractionate the cell into its organelles and generate individual databases for each subcellular component. This has the added advantage of assigning a cellular location to each protein identified. Here we report the development of a two-dimensional gel electrophoresis database of lysosomal proteins.

Amino Acid Sequence↗

PubMed1998

Characterization of rat brain stathmin isoforms by two-dimensional gel electrophoresis-matrix assisted laser desorption/ionization and electrospray ionization-ion trap mass spectrometry.

Stathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two-dimensional electrophoresis (2-DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen-activated protein (MAP), cdc2 kinase, protein kinase A, and Ca2+/calmodulin-dependent kinase-Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post-translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2-DE with apparent molecular weight (Mr)/isoelectric point (pI) values of 15,500/6.2, 15,000/6.1, and 15,000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix-assisted laser desorption/ionization mass spectrometry and electrospray-ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38; however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the Mr/pI locus of this region of the 2-DE gel map. These include: phosphatidyl ethanolamine binding protein (Mr approximately 18,000/pI 6.0), synuclein forms 2 and 3 (Mr approximately 14,000/pI 5.4), and synuclein form 2 (Mr approximately 15,000/pI 5.0).

Amino Acid Sequence↗

PubMed1998

Capillary column chromatography improves sample preparation for mass spectrometric analysis: complete characterization of human alpha-enolase from two-dimensional gels following in situ proteolytic digestion.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in-gel and/or on-membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn (< 350 microm ID) reversed-phase high performance liquid chromatography (RP-HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J., J. Chromatogr. 1992, 599, 119-130). Here we evaluate various capillary column RP-HPLC/mass spectrometric approaches for identifying and characterizing 2-DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused-silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 microm spherical, 300 A pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for sample cleanup prior to protein identification by peptide-mass fingerprinting using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry. While marginally more peptide mass information can be obtained by stepped elution of the peptide mixture with increasing concentrations of organic solvent, best results were obtained by fractionation of the peptide mixture using a linear 60 min gradient. One salient feature of this study was the observation that, in contrast to the stepped elution and gradient approaches, the ionization of peptide T1 (m/z 2402.2 SGETEDTFIADLVV(PeCys)TGQIK) was almost completely suppressed using the ON/OFF approach. Maximal amino acid sequence coverage, a necessary prerequisite for complete characterization of a protein, was accomplished using a capillary column (0.2 mm ID) directly coupled with an electrospray ionization (ESI) ion-trap tandem mass spectrometer. For example, from an in situ tryptic digest of alpha-enolase isolated by 2-DE from the human breast carcinoma cell line MDA-MB231, 71% of the amino acid sequence was obtained. In addition to identifying two possible N-terminal acetylated alpha-enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process-induced modifications of alpha-enolase such as methionine oxidation and cysteine amidoethylation.

Amino Acid Sequence↗

PubMed1998

Two-dimensional mapping and microsequencing of lysosomal proteins from human placenta.

Lysosomes degrade a wide range of macromolecules to yield monomer products which are exported out of the lysosome by a series of transporters. In addition, lysosomes perform a range of other functions which are cell or tissue specific. In order to gain insight into the tissue specific role of lysosomes, carrier-ampholyte two-dimensional electrophoresis (2-DE) was used in combination with N-terminal sequencing to identify the major proteins present in both the membrane and luminal space of placental lysosomes. From the 45 N-terminal peptide sequences generated, 14 luminal and five membrane proteins were identified while three other sequences were novel. The sequenced proteins were a mixture of lysosomal and non-lysosomal proteins. The lysosomal proteins consisted of gamma-interferon-inducible protein (IP-30), Saposin D, cathepsins B and D, beta-hexosaminidase, palmitoyl protein thioesterase, alpha-glucosidase, and LAMP-1. The non-lysosomal proteins were serum albumin, serotransferrin, haemoglobin gamma G chain, alpha-1-antitrypsin, placental lactogen, endoplasmin, peptide binding protein 74, p60 lymphocyte protein, p450 side chain cleavage enzyme and placental alkaline phosphatase. The 2-DE maps obtained in this study are the first to identify the major proteins in both the lumen and membrane of placental lysosomes through sequence analysis, and thus provide the basis upon which to build a complete 2-DE database of the lysosome. Furthermore, the identities of the proteins sequenced from the placental lysosomes suggest a role for lysosomes in the transport of nutrients across the trophoblastic layer.

Adult↗

PubMed1998