| 1. |
- Grubb, A O, et al.
(författare)
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Isolation of human complex-forming glycoprotein, heterogeneous in charge (protein HC), and its IgA complex from plasma. Physiochemical and immunochemical properties, normal plasma concentration
- 1983
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 258:23, s. 707-14698
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Tidskriftsartikel (refereegranskat)abstract
- Human complex-forming glycoprotein, heterogeneous in charge (protein HC) has previously been isolated from urine and immunochemically shown to be present in low and high molecular weight forms in blood plasma (Tejler, L., and Grubb, A. O. (1976) Biochim. Biophys. Acta 439, 82-94). In the present work, the major low and high molecular weight forms of the protein were isolated from plasma by immunosorption followed by gel chromatography. The plasma low molecular weight protein HC and the urinary protein had similar, if not identical, molecular weight, amino acid composition, NH2-terminal and carboxyl-terminal amino acid sequences and electrophoretic mobility. The low molecular weight plasma protein HC carried a yellow chromophore like the urinary protein, but its molar extinction coefficient at 280 nm was lower and its charge heterogeneity less pronounced than that of urinary protein HC. The plasma high molecular weight protein HC had a hydrodynamic volume which was greater than that of monomeric IgA but smaller than that of dimeric IgA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated high molecular weight protein followed by electrophoretic blotting and immunochemical analysis demonstrated that the protein contained four polypeptide chains: two light immunoglobulin chains (Mr = 23,000), one IgA alpha-chain (Mr = 54,000), and one chain with Mr approximately 90,000 which carried both alpha-chain and protein HC antigenic determinants. Whether the protein HC X IgA complex is a functionally significant part of the humoral immune system cannot be decided without further experimentation, but the complex was found to be completely absent from the blood plasma of patients with a selective deficiency of IgA-secreting immunocytes. The isolated low and high molecular weight plasma protein HC components were used as standard proteins in the construction of a quantitative crossed immunoelectrophoretic assay for the simultaneous quantitation of the two major protein HC components in blood plasma. The plasma concentrations of the low and high molecular weight protein HC components were measured by this method in 13 healthy Caucasians. The results for the low molecular weight protein HC were: mean, 20.3 mg/liter, S.D., 3.2 mg/liter, range, 13.6-26.0 mg/liter; and for the protein HC X IgA complex: mean, 293 mg/liter, S.D., 176 mg/liter, range, 36-620 mg/liter.
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| 2. |
- Haataja, Sauli, et al.
(författare)
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Oligosaccharide-receptor interaction of the Galα1-4Gal binding adhesin of Streptococcus suis : Combining site architecture and characterization of two variant adhesin specificities
- 1994
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 269:44, s. 27466-27472
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Tidskriftsartikel (refereegranskat)abstract
- The sugar binding specificities of two groups of Streptococcus suis, a pig pathogen that causes meningitis also in man, were determined. Both the group represented by a recently characterized strain inhibitable by galactose and N-acetylgalactosamine (type PN) and the group inhibitable by galactose (type PO) were found by hemagglutination and solid-phase binding inhibition experiments to recognize the disaccharide Galα1-4Gal of the P1 and Pk blood group antigens. Both types preferred the disaccharide in terminal position. PN showed some, whereas PO showed almost no, binding to the globoside oligosaccharide containing an additional GalNAcβ1-3 residue. The complete hydrogen bonding patterns were determined by using deoxy and other synthetic derivatives of the receptor disaccharide, and the constructed models of the interactions were compared with that of Escherichia coli PapG396 adhesin. The essential hydroxyls for binding were the HO-4', HO- 6', HO-2, and HO-3 hydroxyls on the β'α-side of the Galα1-4Gal molecule. Type PO adhesin also formed weak interactions with the hydroxyls HO-6 and HO-3'. The mechanism differed from that of E. coli, which binds to a cluster of five hydroxyls (HO-6, HO-2', HO-3', HO-4', and HO-6') and thus to a different part of the receptor disaccharide. These results represent the first example of the comparison of the saccharide receptor hydrogen bonding patterns of two bacterial organisms of different origin and show that the same saccharide may be recognized by two different binding mechanisms.
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| 3. |
- Hillarp, A, et al.
(författare)
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Novel subunit in C4b-binding protein required for protein S binding
- 1988
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 263:25, s. 64-12759
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Tidskriftsartikel (refereegranskat)abstract
- C4b-binding protein (C4BP) is a multimeric protein with regulatory functions in the complement system. It also interacts with vitamin K-dependent protein S, which is involved in the regulation of the coagulation system. It has been demonstrated that C4BP consists of seven disulfide-linked, identical 70-kDa subunits, which are arranged to give the molecule a spider-like structure. We now have evidence for the presence of a new subunit in C4BP. On sodium dodecyl sulfate-poly-acrylamide gel electrophoresis it appears as a weakly stainable band with a molecular weight of approximately 45,000. The subunit was isolated by gel filtration in 6 M guanidine hydrochloride of reduced and carboxymethylated C4BP. Its amino-terminal sequence is distinct from previously known protein sequences. The stoichiometry of 45- to 70-kDa subunits was estimated to be 1:9, indicating the presence of one 45-kDa subunit per C4BP molecule. The new subunit was demonstrated to be a disulfide-linked component of the central core of C4BP. It was sensitive to proteolysis by chymotrypsin, and when cleaved the protein S binding ability of C4BP was lost. With protein S bound to C4BP, the 45-kDa subunit was protected from degradation by chymotrypsin, and the protein S binding site remained intact. These data suggest that the new subunit is directly involved in protein S binding.
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| 4. |
- Hillarp, A, et al.
(författare)
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The human C4b-binding protein beta-chain gene
- 1993
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 268:20, s. 23-15017
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Tidskriftsartikel (refereegranskat)abstract
- Human complement component C4b-binding protein (C4BP) is composed of seven alpha-chains and one beta-chain. The alpha- and beta-chains are homologous and both contain multiple copies of short consensus repeats (SCR) and in addition carboxyl-terminal non-repeat regions. Each of the alpha-chains contains a binding site for C4b, whereas the beta-chain binds protein S, a vitamin K-dependent protein involved in the regulation of blood coagulation. The alpha- and beta-chain genes are closely linked in the regulators of complement activation gene cluster on the long arm of human chromosome 1, band 1q32. The human beta-chain gene which has now been characterized was found to span more than 10 kilobases of DNA. The presence of at least two different beta-chain gene transcripts was suggested by the isolation of two new cDNA clones which contained different sequences in their extended 5'-untranslated regions. Northern blot analysis demonstrated that the two clones represented distinct beta-chain mRNAs with different 5' end sequences. One class of beta-chain mRNA (denoted A19) was found to be encoded by six exons and primer extension, and S1 nuclease protection assays revealed multiple closely spaced transcription start sites for this mRNA class. Its 5'-untranslated region and signal peptide was encoded by the first exon. The second class of mRNA (denoted A12) had a different transcription start site and its 5'-untranslated region was derived from at least three exons out of which the last one was formed by utilization of an acceptor splice site within the first A19 exon. Exons encoding the mature beta-chain and the 3'-untranslated region were common to both classes of mRNA. The beta-chain contains three SCRs, out of which the first and second are encoded by individual exons, whereas two exons encode the third SCR. The exon encoding the carboxyl-terminal part of the third SCR also encodes 14 amino acids of the non-repeat region. The last exon encodes the remaining 46 carboxyl-terminal amino acids and the entire 3'-untranslated region. The elucidation of the organization of the beta-chain gene provides insight into the sophisticated molecular structure of C4BP and a basis for future structural and functional studies.
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| 5. |
- Hillarp, A, et al.
(författare)
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The protein S-binding site localized to the central core of C4b-binding protein
- 1987
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 262:23, s. 7-11300
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Tidskriftsartikel (refereegranskat)abstract
- Human C4b-binding protein (C4BP) is a regulator of the classical pathway of the complement system. It appears in two forms in plasma, as free protein and in a noncovalent complex with the vitamin K-dependent coagulation protein, protein S. In the electron microscope C4BP has a spider-like structure with a central core and seven extended tentacles, each of which has a binding site for C4b, although the protein S-binding site has not been unequivocally pinpointed. C4BP was subjected to chymotrypsin digestion which yielded two major fragments, one of 160 kDa representing the central core, and one of 48 kDa representing the cleaved-off tentacles. We have now localized the protein S-binding site to the 160-kDa central core fragment. Using immunoblotting with a panel of polyclonal antisera, the isolated central core was shown to be completely devoid of 48-kDa fragments. The protein S-binding site was susceptible to proteolysis by chymotrypsin, but was protected by a molar excess of protein S included during the proteolysis. The 160-kDa central core fragment consisted of identical, disulfide-linked 25-kDa peptides and a proper disulfide bond arrangement was crucial to protein S binding. Using a direct binding assay it was shown that the isolated central core had the same affinity for protein S as intact C4BP.
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| 6. |
- Schwalbe, Ruth, et al.
(författare)
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Assembly of protein S and C4b-binding protein on membranes
- 1990
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 265:27, s. 16074-16081
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Tidskriftsartikel (refereegranskat)abstract
- The interaction of protein S with membranes and subsequent combination with complement C4b-binding protein (C4BP) was studied. Protein S interacted with phospholipid vesicles in a calcium-dependent manner typical of other vitamin K-dependent proteins. Association of C4BP with protein S showed no apparent selectivity for membrane-bound or solution phase protein S. When bound to the membrane, the protein complexes projected out from the vesicle surface and induced vesicle radius changes of 11.4 nm for tightly packed protein S alone and 17.5 nm for the protein S-C4BP complex. Due to a low density of the protein S-C4BP on the membrane at saturation, the actual projection of this complex out from the membrane surface would be much greater than 17.5 nm. A low saturation density suggested that the protein complex had a large two-dimensional hydrodynamic radius in the plane of the membrane that prevented tight packing of protein. In the presence of calcium, the protein-protein interaction was rapid (ka greater than or equal to 1.10(6) M-1 s-1) and had very high affinity (KD less than or equal to 10(-10) M). The dissociation rate was slow with an estimated rate constant of less than or equal to 2.10(-4) s-1 at 25 degrees C. Protein-protein interaction was much slower in the absence of calcium with an estimated association rate constant of only 2.10(4) M-1 s-1. Consequently, the protein-protein interaction was greatly enhanced by calcium. The very high affinity interaction between protein S and C4BP suggested specificity and an important function for the protein S-C4BP complex in blood. In this regard it was important that C4BP which was bound to protein S on the phospholipid surface could interact with complement protein C4b. These results suggested that protein S may serve an important role in localizing C4BP to negatively charged phospholipid. This would provide regulation of complement activation at sites where the coagulation system is activated such as on the surface of activated platelets.
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| 7. |
- Abraham, Elena T., et al.
(författare)
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Collagen's primary structure determines collagen:HSP47 complex stoichiometry
- 2021
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258. ; 297:6
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Tidskriftsartikel (refereegranskat)abstract
- Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.
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| 8. |
- Andersson, Fredrik, 1977, et al.
(författare)
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Structure and function of a novel type of ATP-dependent Clp protease.
- 2009
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Ingår i: The Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 284:20, s. 13519-32
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Tidskriftsartikel (refereegranskat)abstract
- The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. The main constitutive Clp protease in photosynthetic organisms has evolved into a functionally essential and structurally intricate enzyme. The model Clp protease from the cyanobacterium Synechococcus consists of the HSP100 molecular chaperone ClpC and a mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We have purified the ClpP3/R complex, the first for a Clp proteolytic core comprised of heterologous subunits. The ClpP3/R complex has unique functional and structural features, consisting of twin heptameric rings each with an identical ClpP3(3)ClpR(4) configuration. As predicted by its lack of an obvious catalytic triad, the ClpR subunit is shown to be proteolytically inactive. Interestingly, extensive modification to ClpR to restore proteolytic activity to this subunit showed that its presence in the core complex is not rate-limiting for the overall proteolytic activity of the ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable similarities to the 20 S core of the proteasome, revealing a far greater degree of convergent evolution than previously thought between the development of the Clp protease in photosynthetic organisms and that of the eukaryotic 26 S proteasome.
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| 9. |
- Awad, W., et al.
(författare)
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Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:38, s. 22991-23008
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Tidskriftsartikel (refereegranskat)abstract
- Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the Cterminus and also the topology of Gpc1 with respect to the membrane. The Cterminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation.
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| 10. |
- Azarkina, Natalia, et al.
(författare)
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A bb´-type quinol oxidase in Bacillus subtilis strain 168
- 1999
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 1083-351X .- 0021-9258. ; 274, s. 32810-32817
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Tidskriftsartikel (refereegranskat)abstract
- The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa3, which is a cytochrome c oxidase, and cytochrome aa3 and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa3 and caa3 terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a 'cytochrome o'-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence or B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'- type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b558b595b'.
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