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K K Stanley

Publications and source records attributed to K K Stanley.

At least 19 recordsLinked to original sources
PubMed1992

A monoclonal anti-peptide antibody reacting with the insulin receptor beta-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors.

A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.

Amino Acid Sequence↗
PubMed1992

High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors.

Analysis of untreated fresh blood plasma from healthy, fasting donors revealed that high density lipoprotein (HDL) particles carry most (approximately 85%) of the detectable oxidized core lipoprotein lipids. Low density lipoprotein (LDL) lipids are relatively peroxide-free. In vitro the mild oxidation of gel-filtered plasma from fasting donors with a low, steady flux of aqueous peroxyl radicals initially caused preferential oxidation of HDL rather than LDL lipids until most ubiquinol-10 present in LDL was consumed. Thereafter, LDL core lipids were oxidized more rapidly. Isolated lipoproteins behaved similarly. Preferential accumulation of lipid hydroperoxides in HDL reflects the lack of antioxidants in most HDL particles compared to LDL, which contained 8-12 alpha-tocopherol and 0.5-1.0 ubiquinol-10 molecules per particle. Cholesteryl ester hydroperoxides (CEOOHs) in HDL and LDL were stable when added to fresh plasma at 37 degrees C for up to 20 hr. Transfer of CEOOHs from HDL to LDL was too slow to have influenced the in vitro plasma oxidation data. Incubation of mildly oxidized LDL and HDL with cultured hepatocytes afforded a linear removal of CEOOHs from LDL (40% loss over 1 hr), whereas a fast-then-slow biphasic removal was observed for HDL. Our data show that HDL is the principal vehicle for circulating plasma lipid hydroperoxides and suggest that HDL lipids may be more rapidly oxidized than those in LDL in vivo. The rapid hepatic clearance of CEOOHs in HDL could imply a possible beneficial role of HDL by attenuating the build-up of oxidized lipids in LDL.

Adult↗
PubMed1992

Isolation of sequences from a random-sequence expression library that mimic viral epitopes.

We describe the use of random peptide sequences for the mapping of antigenic determinants. An oligonucleotide with a completely degenerate sequence of 17 or 23 nucleotides was inserted into a bacterial expression vector. This resulted in an expression library producing random hexa- or octapeptides attached to a beta-galactosidase hybrid protein. Mimotopes, or antigenic sequences that mimic an epitope, were selected by immunoscreening of colonies with monoclonal antibodies, which were specific for antigenic sites on the spike protein of the coronavirus transmissible gastroenteritis virus. We report one mimotope for antigenic site II, eight for site III and one for site IV. The site III and site IV mimotopes were closely similar to the corresponding linear epitopes, localized previously in the amino acid sequence of the S protein. An alignment of the site II mimotope and the sequence of the S protein around Trp97, which is substituted in escape mutants, suggests that this mimotope mimics a conformational epitope located around residues 97-103. Applications of mimotopes to epitope mapping, serodiagnosis and vaccine development are discussed.

Amino Acid Sequence↗
PubMed1991

pUBEX/pUBSEX: a versatile expression vector system for production of fusion and nonfusion proteins in Escherichia coli.

Despite the large number of expression vectors now available, none provide the facility of allowing fusion and nonfusion protein production from the same vector system. In some situations it is preferable to obtain an insoluble fusion protein, in others a soluble nonfusion protein may be required. We have designed, constructed and tested a modification of the pEX vectors, in which it is possible to express the product of a suitably inserted cDNA either as part of a Cro-beta-galactosidase (Cro-beta Gal) fusion or as a delta Cro fusion which contains only nine noninsert-encoded amino acids at its N terminus. The conversion from Cro-beta Gal to delta Cro fusion protein production is achieved by a simple intramolecular deletion of lacZ sequence from the pUBEX vector, to create the pUBSEX variant. Plasmid pUBEX can be induced to produce large amounts of insoluble Cro-beta Gal fusion proteins, whereas pUBSEX will produce predominantly soluble delta Cro fusion proteins.

Animals↗
PubMed1990

Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38).

Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network.

Amino Acid Sequence↗
PubMed1990

Identification of epitopes on the human insulin receptor reacting with rabbit polyclonal antisera and mouse monoclonal antibodies.

The sequential epitopes on the human insulin receptor recognized by polyclonal and monoclonal antibodies were investigated using a recombinant DNA technique. Short random fragments of receptor cDNA were cloned and expressed in Escherichia coli as beta-galactosidase fusion proteins by using the expression vector pUEX1. Immunoreactive peptides were detected by colony blotting and identified by sequencing the corresponding cDNA inserts. Eleven antigenic determinants were located with rabbit antisera, nine of these being on the alpha-subunit and two on the beta-subunit of which one was intracellular. Two human autoantibodies reacted with the alpha-subunit on blots, but no sequential epitopes could be located. In the rabbit sera, antibody reacting with these linear epitopes represented a substantial fraction (approximately 50%) of antibody reacting with reduced denatured receptor on blots, but a generally smaller fraction (5-40%) of antibody reacting with solubilized native receptor. Epitope-specific subfractions of antibodies were purified by binding to an elution from bacterial fusion proteins. All of these subfractions reacted with denatured receptor on nitrocellulose blots, but only three precipitated native receptor (epitopes between amino acids 190 and 231, 654 and 669, 954 and 982) and none inhibited insulin binding. (The numbering system used in this manuscript is that of Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf. L., Clauser, E., Ou, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., and Rutter, W. J. (1985) Cell 40, 747-758). The binding sites of two monoclonal antibodies were also determined. One of these antibodies (83-14) is insulin-mimetic, but inhibits insulin binding and its epitope on the alpha-subunit (between amino acids 469 and 592) may contribute to the insulin binding site in the folded protein. The other antibody (18-44) binds close to the N terminus of the beta-subunit (amino acids 765-770) and does not inhibit insulin binding, but does mimic insulin action. The identification of epitopes therefore provides information on receptor conformation and allows structural domains to be identified which are involved in the functional effects of different antibodies.

Amino Acid Sequence↗
PubMed1990

A new recombinant DNA strategy for the molecular cloning of rare membrane proteins.

We have constructed a cDNA library in the plasmid expression vector pUEX enriched in sequences encoding membrane proteins. The procedure involved positive selection of sequences common to two different rat tissues (thus excluding tissue-specific mRNA) followed by positive selection between this material and RNA extracted from membrane bound polysomes (thus excluding cytoplasmic proteins). The resultant library prepared from rat kidney cDNA hybridized with rat liver poly(A)+ RNA, contained 30,000 clones and was shown to be enriched in cDNAs encoding membrane proteins. Seventeen clones selected because they encode large fusion proteins were shown to be single copy in the library, and not present in nucleotide data banks. Thus the strategy is particularly suitable for cloning low abundance cDNAs encoding membrane proteins.

Animals↗
PubMed1989

Structure of the low-density lipoprotein receptor-related protein (LRP) promoter.

The low-density Lipoprotein receptor-related protein (LRP) is a 4544-amino-acid membrane protein which closely resembles the LDL receptor in its arrangement of cysteine-rich motifs. Binding studies have suggested that one function of the molecule is as a receptor for ligands containing apolipoprotein E. We present here the sequence and structure of the promoter region of the LRP. These data show that the LRP contains no sterol regulatory element, and is not down-regulated by sterols like the LDL receptor. This lends further support to the identity of the LRP as a chylomicron remnant receptor.

Animals↗
PubMed1989

Sulfation of two tyrosine-residues in human complement S-protein (vitronectin).

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.

Amino Acid Sequence↗
PubMed1989

The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein.

The low-density-lipoprotein (LDL) receptor is a cell-surface protein that plays an important part in the metabolism of cholesterol by mediating the uptake of LDL from plasma into cells. Although LDL particles bind to the LDL receptor through their apolipoprotein B (apo B) and apolipoprotein E (apo E) moieties, other apo E-containing particles, like chylomicron remnants, are not dependent on the LDL receptor for uptake into cells. Chylomicrons formed in the intestinal mucosa during the absorption of the products of digestion, are processed by the peripheral circulation by lipoprotein lipase, which catalyses the breakdown of triglycerides in chylomicrons to free fatty acids and glycerol. The resulting chylomicron remnants, which are cholesterol-rich lipoproteins, are subsequently taken up in the liver. A second distinct protein that binds to apo E-containing lipoproteins, but not to LDL, has been proposed to be the receptor mediating the clearance of chylomicron remnants from the plasma. This protein has a relative molecular mass (Mr) of 56,000 (56K). More recent studies have failed, however, to establish whether this protein is a cell-surface receptor. Here we describe crosslinking experiments in which apo E liposomes were found to bind specifically to the cell surface of hepG2 cells and to human liver membranes. The size and immunological cross-reactivity of the protein to which the liposomes bound was indistinguishable from that of the recently cloned and sequenced LDL-receptor-related protein, LRP. We therefore conclude that the LRP might function as an apo E receptor.

Animals↗
PubMed1989

Intracellular targetting signals of polymeric immunoglobulin receptors are highly conserved between species.

A rat liver cDNA library, constructed in the plasmid expression vector pUEX, was immunoscreened using a rabbit polyclonal antiserum raised against rat liver Golgi membrane proteins. A sub-set of isolated clones were shown to encode the rat polymeric immunoglobulin receptor (pIgR). DNA sequence analysis of these clones provided the complete coding sequence of rat pIgR. Subsequent alignment of rat, rabbit and human predicted amino acid sequences demonstrated that the greatest degree of homology between the three pIgRs lies in their cytoplasmic tails; a region previously shown to be important for correct targetting and trancytosis of rabbit pIgR [(1984) Nature 308, 37-43].

Amino Acid Sequence↗
PubMed1989

Complement C9 is inserted into membranes in a globular conformation.

Complement component C9 undergoes a major conformational change during its insertion into a biological membrane from a globular to an extended form. At 0 degrees C a single C9 binds but a membrane attack complex (MAC) is not formed. We show that the C9 bound at 0 degrees is accessible to the intracellular space and sensitive to trypsin digestion, suggesting that C9 inserts in its globular state and requires an elevated temperature in order to change conformation.

Animals↗
PubMed1988

Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor.

We describe a cell surface protein that is abundant in liver and has close structural and biochemical similarities to the low density lipoprotein (LDL) receptor. The complete sequence of the protein containing 4544 amino acids is presented. From the sequence a remarkable resemblance to the LDL-receptor and epidermal growth factor (EGF) precursor is apparent. Three types of repeating sequence motifs entirely account for the extracellular domain of the molecule. These are arranged in a manner resembling four copies of the ligand binding and the EGF-precursor homologous region of the LDL-receptor. Following a proline-rich segment of 17 amino acids are found six consecutive repeats with close homology to EGF. A single membrane-spanning segment precedes a carboxy-terminal 'tail' of 100 amino acids. This contains two seven-amino acid sequences with striking homology to the cytoplasmic tail of the LDL-receptor in the region that contains the signal for clustering into coated pits. The mRNA for this protein is most abundant in liver, brain and lung. By using an antibody raised against a 13-amino acid peptide corresponding to the deduced amino acid sequence of the carboxy-terminus of the protein we have demonstrated its existence on the cell surface and its abundance in liver. Like the LDL-receptor this protein also strongly binds calcium, a cation absolutely required for binding of apolipoproteins B and E to their receptors. We propose that this LDL-receptor related protein (LRP) is a recycling lipoprotein receptor with possible growth-modulating effects.

Amino Acid Sequence↗
PubMed1988

Relationships between the gene and protein structure in human complement component C9.

Human complement component C9 is a multidomain protein for which a large number of surface topographical features have been determined. We have analyzed the exon-intron boundaries of the human C9 gene and find a good correlation between splice sites and surface features of the protein but little correlation with the putative protein domain structure, even in the cysteine-rich sequence homology with the low-density lipoprotein (LDL) receptor which is likely to be an independently folded structural motif. This is surprising because in the LDL receptor the same sequence is precisely bounded by introns, and it has been assumed that this sequence is present in both proteins as a result of exon shuffling. We deduce that substantial rearrangement of the exon-intron structure of the C9 gene must have occurred before the exchange of cysteine-rich domains, possibly linked to the process of exon duplication which was required to generate the repeats in the LDL receptor.

Amino Acid Sequence↗
PubMed1988

cDNA cloning of granzyme C, a granule-associated serine protease of cytolytic T lymphocytes.

A cDNA clone corresponding to the complete amino acid sequence of a putative protease CCP2 of murine cytotoxic T lymphocytes was isolated and sequenced. The clone encodes a 248-residue long serine esterase. The deduced N-terminal amino acid sequence is identical over 40 residues to that of granzyme C, a protease of unknown function present in granules of cytotoxic lymphocytes. Analysis of the sequence of granzyme C/CCP2 reveals high homology to other granzyme proteases, i.e. granzyme A (40%) and granzyme B (67%) and to rat mast cell protease II (46%). The amino acids lining the specificity pocket are well conserved between granzyme B, C, and rat mast cell protease II, but not granzyme A, suggesting a similar general specificity of these three proteases.

Amino Acid Sequence↗
PubMed1988

Sequence homology of complement C8 gamma chain with alpha 1-microglobulin and its implications for C8 structure and function.

Complement C8 gamma is a disulphide bonded subunit of C8 with no known homology or function. We show here that it strongly resembles alpha 1-microglobulin and protein HC in both length and sequence suggesting a common genetic origin. By extrapolation from the alpha 1-microglobulin family we are able to suggest that the cysteine residue of C8 gamma is involved in disulphide bonding to C8 alpha, and a possible physiological role in the regulation of the inflammatory response. Comparison of C8 gamma with C8 alpha shows that one region of C8 alpha bears a structural homology with the region of C8 gamma containing the free cysteine residue. This homology includes a cysteine residue of C8 alpha, for which no equivalent is found in C8 beta or C9, and which is therefore a likely candidate for C8 gamma attachment.

Alpha-Globulins↗