Endo180 binds to the C-terminal region of type I collagen

Type I collagen is a fibril-forming heterotrimer composed of two alpha1 and one alpha2 chains and plays a crucial role in cell-matrix adhesion and cell differentiation. Through a comprehensive differential display screening of oncogenic ras target genes, we have shown that the alpha1 chain of type I collagen (col1a1) is markedly down-regulated by the ras oncogene through the mitogen-activated protein kinase pathway. Although ras-transformed cells are no longer able to produce and secrete endogenous collagen, they can still adhere to exogenous collagen, suggesting that the cells express a collagen binding factor(s) on the cell surface. When the region of col1a1 encompassing the C-terminal glycine repeat and C-prodomain (amino acids 1000-1453) was affinity-labeled with human placental alkaline phosphatase, the secreted trimeric fusion protein could bind to the surface of Ras-transformed cells. Using biochemical purification followed by matrix-assisted laser desorption/ionization mass spectrometry analysis, we identified this collagen binding factor as Endo180 (uPARAP, CD280), a member of the mannose receptor family. Ectopic expression of Endo180 in CosE5 cells followed by in situ staining and quantitative binding assays confirmed that Endo180 indeed recognizes and binds to placental alkaline phosphatase. The interaction between Endo180 and the C-terminal region of type I collagen appears to play an important role in cell-matrix adhesion.     

The leader region of pre-maltose binding protein binds amphiphiles. A model for self-assembly in protein export

Maltose binding protein, like most periplasmic proteins, is resistant to a variety of proteinases. Treatment of pre-maltose binding protein with trypsin, chymotrypsin, or proteinase K removes an amino-terminal domain of the same approximate size as the leader sequence without degrading the mature portion of the protein. In addition, pre-maltose binding protein is as active as mature in binding maltose (Ferenci, T., and Randall, L.L. (1979) J. Biol. Chem. 254, 9979-9981). By these criteria, the precursor and mature proteins are in the same conformation except for the exposed leader sequence on the precursor. We have compared the ability of these proteins to interact with amphipaths, such as detergents. The precursor protein binds to Triton X-100, while the mature protein does not. We propose that the leader domain is responsible for detergent binding. Mutations in the leader region of the precursor which block export in vivo prevent detergent binding in vitro. A mutant with a mild export defect can still bind detergent. This correlation between detergent binding by precursors with related leaders and export efficiency of each precursor suggests that hydrophobic partition of the leader may initiate pre-protein transfer across the membrane.     

Fibronectin binds to amyloid P component. Localization of the binding site to the 31,000 dalton C-terminal domain

Fibronectin has been shown to play an important role in reticuloendothelial system functioning as well as in neutrophil and fibroblast migration to tissue injury sites. Fibronectin binds several macromolecules including components of the acute phase response. We have studied the interaction of fibronectin with the amyloid P component (AP). This glycoprotein, closely related to C-reactive protein, is deposited together with amyloid fibrils and is also a normal constituent of human fibronectin, its whole tryptic digest, and isolated fragments; fibronectin was retained by immobilized AP in a molar ratio fibronectin:AP of 1:5.8. In this paper we localized the binding site for AP in a tryptic 31 kDa fragment, near the C-terminal end of the fibronectin molecule. A shorter fragment of 22 kDa starting at position 82 of the 31 kDa domain and containing all the disulfide bridges present in the 31 kDa domain did not bind to AP; therefore the active site appears to be located within the 81 N-terminal residues of the 31 kDa fragment. To further support this conclusion, reduction and alkylation of either fibronectin or the 31 kDa fragment had no effect on their binding properties.     

Protein Z-dependent protease inhibitor binds to the C-terminal domain of protein Z

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K(D)) of approximately 7 nm. However, the apparent K(D) for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes approximately 6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.     

基质Gla蛋白在大鼠附睾发育过程中的表达及定位

目的明确基质Gla蛋白(matrix Gla protein,MGP)在大鼠附睾发育过程中的表达特征。方法采用实时定量PCR和免疫荧光染色方法,对MGP在大鼠附睾不同发育阶段的表达及定位进行检测。结果实时定量PCR结果显示,MGP mRNA在6d、10d、3w、5w、7w、8w、10w和12w的大鼠附睾中均有表达,其表达量在3w达到最高峰,3w至8w表达量逐渐降低,成年大鼠(10～12w)MGP的表达量逐渐升高并稳定在较高水平。免疫荧光染色显示MGP在10d、3w的大鼠附睾各个节段均有表达,在7w、12w的表达主要集中于大鼠附睾体部和尾部,且MGP定位于附睾上皮主细胞和亮细胞。结论MGP在大鼠附睾发育的关键分化期高表达,成年后主要定位于附睾体部和尾部的主、亮细胞,可能对附睾的形态发育和管腔钙稳态的维持起重要作用。     

The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5'-incision.

The incisions in the DNA at the 3'- and 5'-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC. The exact requirements for the two incision events, however, are different. It has previously been shown that the 3'-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC. This interaction, however, is dispensable for the 5'-incision. Here we show that the C-terminal domain of the UvrC protein is essential for the 5'-incision, whereas this domain can be deleted without affecting the 3'-incision. The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5'-incision reaction in human nucleotide excision repair. Both in the UvrC and the ERCC1 domain a Helix-hairpin-Helix (HhH) motif can be indicated, albeit at different positions. Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding. In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA. Analysis of protein-DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC-DNA complex after the 3'-incision has taken place. We propose that after the initial 3'-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5'-incision reaction.     

Matrix Gla protein is differentially expressed during the deposition of a calcified matrix by vascular pericytes

PCR-based subtractive hybridisation was used to identify genes up-regulated when pericytes undergo osteogenic differentiation and deposit a calcified matrix. cDNA pools were generated from confluent pericytes and from pericyte cultures containing calcified nodules. A pericyte cDNA library was screened with the product of the subtraction procedure (calcified minus confluent cDNA) and the majority of the positive clones were identified as matrix Gla protein (MGP). Northern analysis and immunohistochemistry demonstrated that MGP was only expressed by pericytes in calcified nodules. Antibodies to MGP inhibited the deposition of a calcified matrix by pericytes, suggesting that MGP regulates both cell differentiation and calcification.     

Kynurenine binds to the peptide binding region of the chaperone alphaB-crystallin

UV filters, such as kynurenine, are present in the human lens. They are spontaneously unstable at neutral pH and deaminate to form reactive alpha, beta unsaturated ketones. This process becomes more prominent after the lens barrier develops in middle age. Here we show that deaminated kynurenine reacts primarily with histidine residues in alphaB-crystallin: a major lens protein that lacks cysteine. Five of the nine histidines in alphaB-crystallin were found to be conjugated with kynurenine. Furthermore, a major site of covalent modification was at histidine 83, which is found in the putative peptide binding region of alphaB-crystallin; a site crucial for its role as a chaperone. We propose that modification of alphaB-crystallin by UV filters may compromise the chaperone action of this protein.     

Apo-calmodulin binds with its C-terminal domain to the N-methyl-D-aspartate receptor NR1 C0 region

Calmodulin (CaM) is the major Ca2+ sensor in eukaryotic cells. It consists of four EF-hand Ca2+ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca2+ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca2+ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca2+ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca2+ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to full-length NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2' domain. The Ca2+ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca2+. Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca2+ binds to CaM.     

Specific binding of plasminogen to vitronectin. Evidence for a modulatory role of vitronectin on fibrin(ogen)-induced plasmin formation by tissue plasminogen activator.

Vitronectin immobilized onto polystyrene microtiter wells was demonstrated to specifically bind plasminogen in a concentration-dependent manner, yielding an estimated KD = 0.4 microM. Heparin only moderately interfered with the vitronectin-plasminogen interaction, whereas high concentrations of 6-amino-hexanoic acid inhibited binding. Utilizing a ligand-blotting procedure in which plasminogen was reacted with proteolytic fragments of vitronectin, transblotted onto nitrocellulose, the plasminogen-binding site of vitronectin was localized to the heparin-binding domain of the adhesive protein. Moreover, vitronectin was found to inhibit in a dose-dependent fashion the fibrin(ogen)-induced activation of plasminogen by tissue plasminogen activator. These results provide the first evidence for a novel vitronectin-mediated control of plasminogen activation potentially relevant for directional clot-lysis and plasmin-dependent proteolysis in extracellular matrices.     

Matrix Gla protein accumulates at the border of regions of calcification and normal tissue in the media of the arterial vessel wall.

Vitamin K-dependent matrix Gla protein (MGP) has been suggested to play a role in the inhibition of soft-tissue calcification. Here we report the expression of recombinant prokaryotic MGP as part of a fusion protein and the preparation of two antibodies that specifically recognize MGP. Monoclonal antibodies were raised against synthetic peptides homologous to the sequences 3-15 and 63-75 of human MGP. Both antibodies recognize recombinant and synthetic human MGP. Immunohistochemical analysis showed that MGP was associated with the extracellular matrix of noncalcified bone and with chondrocytes in cartilage. In the healthy human arterial vessel wall, MGP antigen was demonstrated in association with smooth muscle cells and elastic laminae of the tunica media and with the extracellular matrix of the adventitia. Colocalization with the elastic laminae was lost at sites of medial calcification; in both human and rat arteries, high amounts of MGP were found in the extracellular matrix at borders of intimal and medial calcification. Our data demonstrate the close association between MGP and calcification. It is suggested that undercarboxylated MGP is biologically inactive and that poor vascular vitamin K status may form a risk factor for vascular calcification.     

A metabolite of acetaminophen covalently binds to the 56 kDa selenium binding protein.

Acetaminophen is metabolized by cytochrome P450 to a reactive metabolite that covalently binds to proteins and this binding correlates with the hepatotoxicity. The major protein adduct was previously reported to be a 55 kDa protein that was detected on Western blots using antisera specific for 3-(cystein-S-yl)acetaminophen. In this study, the 55 kDa protein was isolated using a combination of ion exchange fast flow chromatography, hydroxyapatite HPLC and anion exchange HPLC. Amino acid sequences of 8 internal peptides from a trypsin digestion of the 55 kDa protein were found to have 97% homology with the deduced amino acid sequence from a cDNA that corresponds to a 56 kDa selenium binding protein. This is the first report of a specific protein to which a metabolite of acetaminophen covalently binds.