Identification of a bone sialoprotein receptor in osteosarcoma cells

Bone sialoprotein (BSP) is an extracellular matrix glycoprotein associated with the mineral bone matrix. The amino acid sequence of BSP contains an Arg-Gly-Asp (RGD) sequence which confers to the protein cell binding properties (Oldberg, A., Franzén, A., and Heineg?rd, D. (1988) J. Biol. Chem. 263, 19430-19432). When BSP was used as an affinity matrix to isolate a cell surface receptor from rat osteosarcoma cells, a protein composed of polypeptides similar in size to those of a previously characterized vitronectin receptor was obtained. This putative BSP receptor, like the vitronectin receptor, bound also to an affinity matrix made of an RGD-containing heptapeptide. Moreover, similar patterns of inhibition of cell attachment to BSP and vitronectin was obtained with variant RGD-containing peptides, with BSP and with vitronectin. Finally, an anti-vitronectin receptor antiserum immunoprecipitated a receptor identical in size to the receptor bound to a BSP affinity matrix. These results show that BSP is recognized by an RGD-directed receptor and that both vitronectin and BSP can bind to this receptor.     

Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells

The relationship between apoB-100 and the membrane of the endoplasmic reticulum (ER) has been studied by a combination of pulse-chase methodology and subcellular fractionation. HepG2 cells were pulse-labeled with [35S]methionine for 3 min and chased with cold methionine for periods between 0 and 20 min. ApoB-100 and albumin, present in the membrane as well as in the luminal content of the ER vesicles, were isolated after each chase period. The results indicated that apoB-100 was cotranslationally bound to the membrane of the ER, and from this membrane-bound form, was transferred to the lumen after a delay of 10-15 min. Albumin was, as could be expected for a typical secretory protein, cotranslationally sequestered in the lumen of the ER. Apo-B-100-containing lipoproteins present in the microsomal lumen were analyzed by ultracentrifugation in a sucrose gradient. ApoB-100 occurred on rounded particles in three density regions: (i) d 1.1065-1.170 g/ml (Fraction I), (ii) d 1.011-1.045 g/ml (Fraction II), and (iii) d less than 1.011 g/ml (Fraction III). Fraction I, isolated from cells cultured in the absence of oleic acid, contained a homogenous population of particles with a mean diameter of approximately 200 A. Fraction I isolated from cells cultured in the presence of oleic acid was slightly more heterogeneous and had a mean diameter of approximately 250 A. Fractions II and III had mean diameters of 300 and 500 A, respectively. Cholesterol esters and triacylglycerol were the quantitatively dominating lipid constituents of all three fractions. Pulse-chase experiments indicated that Fraction I contained the newly assembled lipoproteins. With increasing chase time, the apoB-100 radioactivity was redistributed from Fraction I to Fractions II and III, indicating that Fraction I is converted into Fractions II and III during the intracellular transfer. Particles corresponding to Fractions II and III were by far the most abundant lipoproteins found in the medium. The results presented support the possibility of a sequential assembly of apoB-100-containing lipoproteins.     

Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function

The glycoprotein hormone erythropoietin (Ep), the primary regulator of erythropoiesis, is synthesized by the kidney and secreted as the mature protein with three N-linked and one O-linked oligosaccharide chains. To investigate the role(s) of each carbohydrate moiety in the biosynthesis and function of Ep, we have used oligonucleotide-directed mutagenesis of a cDNA for human Ep to alter the amino acids at each of the carbohydrate attachment sites. Each mutated cDNA construct was expressed in stably transfected sublines of a kidney cell line, baby hamster kidney. We show, by preventing attachment of N-linked carbohydrate at asparagines 38 or 83, or preventing O-linked glycosylation at serine 126, that glycosylation of each of these specific sites is critical for proper biosynthesis and secretion of Ep. Fractionation of cellular extracts demonstrated that the mutant proteins lacking glycosylation at each of these three sites, (38, 83, and 126) were associated mainly with membrane components or were degraded rapidly. Less than 10% of these three mutant proteins were processed properly and secreted from the cells. The Ep protein lacking N-linked glycosylation at asparagine 24 is synthesized and secreted as efficiently as native Ep. The carbohydrates at positions 24 and 38 may be involved in the biological activity of Ep, since the absence of either of the oligosaccharide side chains at these positions reduced the hormone's biological activity.     

Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.     

Comparison of the amino acid sequences of human protamines HP2 and HP3 and of intermediate basic nuclear proteins HPS1 and HPS2. Structural evidence that HPS1 and HPS2 are pro-protamines

Two intermediate nuclear basic proteins HPS1 and HPS2 were isolated from human sperm. They were characterized by their electrophoretic mobility in acid-urea gels, their amino acid composition, and their peptide maps after digestion by endoproteinase Lys-C and by endoproteinase Glu-C. Their amino-terminal amino acid sequences have also been determined. The structural data thus obtained suggest that HPS1 and HPS2 are precursors of human protamines HP2 and HP3.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.     

Inhibition of bilirubin UDPglucuronosyltransferase activity by triphenylacetic acid and related compounds

Bilirubin UDPglucuronosyltransferase of rat or human liver microsomes was inhibited, in vitro, by triphenylacetic acid and by structurally related arylcarboxylic acids. This inhibition appeared to be competitive towards bilirubin, and mixed-type towards UDPglucuronic acid. A decrease in the number of phenyl rings or the absence of the carboxyl group in the molecule gave structures which did not affect enzyme activity, showing that both the triphenyl moiety and the carboxyl group were necessary for the inhibition. On the other hand, successive additions of methylene groups in the aliphatic chain progressively increased inhibitory potency. Kappi,bilirubin for triphenylacetic acid was 96 microM compared with 5 microM for 7,7,7-triphenylheptanoic acid. The inhibition of bilirubin UDPglucuronosyltransferase was not due to displacement of bilirubin from albumin. On the basis of these results an attempt was made to delineate the molecular events leading to glucuronidation of bilirubin.     

Uptake of the hormone 1,25-dihydroxyvitamin D3 by the dog liver

The hepatic uptake of the hormone 1,25-dihydroxyvitamin D3 has been studied, in vivo, using the multiple indicator dilution technique. The fractional uptake of 1,25-dihydroxyvitamin D3 during a single circulatory passage across the dog liver has been estimated at 34.4 +/- 3.3% while its hepatic clearance was estimated at 364.3 +/- 94.1 mL/min. The hepatic uptake of 1,25-dihydroxyvitamin D3 is discussed in relation to its systemic bioavailability following intravenous or oral administration as well as in relation to the hepatic uptake of other vitamin D sterols; it is postulated that the hepatic uptake of vitamin D sterols does not seem to be mediated by specific receptors on the liver plasma membrane; it seems, however, that the hepatic uptake of vitamin D sterols may be inversely related to their relative affinity for the circulating carrier, the vitamin D binding protein.     

X-ray crystal structure of galabiose, O-alpha-D-galactopyranosyl-(1---4)-D-galactopyranose

O-alpha-D-Galactopyranosyl-(1---4)-D-galactopyranose, C12H22O11, Mr = 342.30, crystallises in the orthorhombic space group P2(1)2(1)2(1), and has alpha = 5.826(1), b = 13.904(3), c = 17.772(4) A, Z = 4, and Dx = 1.579 g.cm-3. Intensity data were collected with a CAD4 diffractometer. The structure was solved by direct methods and refined to R = 0.063 and Rw = 0.084 for 2758 independent reflections. The glycosidic linkage is of the type 1-axial-4-axial with torsion angles phi O-5' (O-5'-C-1'-O-1'-C-4) = 98.1(2) degrees, psi C-3 (C-3-C-4-O-1'-C-1') = -81.9(3) degrees, phi H (H-1'-C-1'-O-1'-C-4) = -18 degrees, and psi H (H-4-C-4-O-1'-C-1') = 35 degrees. The conformation is stabilised by an O-3 . . . O-5' intramolecular hydrogen-bond with length 2.787(3) A and O-3-H . . . O-5' = 162 degrees. The glycosidic linkage causes a folding of the molecule with an angle of 117 degrees between the least-square planes through the pyranosidic rings. The crystal investigated contained 56(1)% of alpha- and 44(1)% of beta-galabiose as well as approximately 70% of the gauche-trans and approximately 30% of the trans-gauche conformers about the exocyclic C-5'-C-6' and C-5-C-6 bonds. The crystal packing is governed by hydrogen bonding that engages all oxygen atoms except the intramolecular acceptor O-5' and the glycosidic O-1' oxygen atoms.