Kimimitsu Oda

Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes

The effects of brefeldin A on intracellular transport and posttranslational modification of complement C3 (C3) were studied in primary culture of rat hepatocytes. In the control culture C3 was synthesized as a pre‐cursor (pro‐C3), which was processed to the mature form with α‐ and β‐subunits before its discharge into the medium. In the presence of brefeldin A the secretion of C3 was strongly blocked, resulting in accumulation of pro‐C3. However, after a prolonged interval the mature form of C3 was finally secreted. The results indicate that brefeldin A impedes translocation of pro‐C3 to the Golgi complex where pro‐C3 is converted to the mature form, but not its proteolytic processing, in contrast to the effects of monensin and weakly basic amines.

Bafilomycin A1 inhibits the targeting of lysosomal acid hydrolases in cultured hepatocytes

Effects of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase, on the synthesis and processing of cathepsin D and cathepsin H were investigated in primary cultured rat hepatocytes. Pulse-chase experiments showed that after being synthesized as procathepsin D and procathepsin H the precursors were converted into mature forms in the control cells as the chase time elapsed. However, in the presence of 5 x 10(-7) M of bafilomycin A1, both precursors were largely secreted into the medium and no mature forms were found within the cells. Thus bafilomycin A1 mimics lysosomotropic amines with regard to perturbation of the targeting of lysosomal acid hydrolases. In contrast, bafilomycin A1 was found not to inhibit processings of proalbumin and procomplement component 3, which are thought to occur at the acidic trans-Golgi, implying that the proteolytic event of the proproteins is not sensitive to an increase of intra-Golgi pH. The results suggest that bafilomycin A1 is useful as a pH-perturbant to study the role of acidity in living cells.

Monensin inhibits the conversion of proalbumin to serum albumin in cultured rat hepatocytes

Abstract Effects of monensin, a carboxylic ionophore, on intracellular transport of albumin were studied in primary cultured rat hepatocytes. The lag time after which newly synthesized albumin first appeared in medium was 10 min in the control cells, while it was prolonged to 40 min in the monensin-treated cells. In addition, this inhibition of secretion by monensin was accompanied by an intracellular accumulation of proalbumin. The results strongly suggest that monensin arrests the intracellular transport of proalbumin before the site where its conversion takes place.

Multiple Forms of Rat-Serum α1-Protease Inhibitor

alpha 1-Protease inhibitor was purified from rat serum by two different methods, of which an immunoaffinity method should be preferentially used to obtain all of the multiple forms. The protein thus obtained showed a single protein band in dodecylsulphate/polyacrylamide gel electrophoresis corresponding to a molecular weight of 54000, and contained 13.2% carbohydrate by weight. By column isoelectric focusing in a pH 3.5-5.0 gradient the purified alpha 1-protease inhibitor was separated into five forms with pI values from 4.3-4.7. The amino acid composition of each form was identical, while sialic acid content was significantly different from each other. The most acidic form contained 6.7 residues/molecule, the most basic form, 5.1 residues/molecule, and three forms between them showed proportionally intermediate values between the two. When alpha 1-protease inhibitor was treated with neuraminidase, the five forms were converted finally into three major forms with pI values of 5.3-5.7. In addition, the major form (band 3) of the inhibitor was also converted into three forms after complete removal of sialic acid. These results suggest that alpha 1-protease inhibitor originally exists as three forms with different charges, possibly due to modification of amino acids which might not be detectable by the amino acid composition analysis in the present study. A possible explanation was presented for involvement of sialic acid in appearance of multiple forms originating from three parental forms.

Inhibitory effect of colchicine on translocation of alkaline phosphatase to the plasma membrane concomitant to its induction in rat liver

A single injection of colchicine (1--3 mg/kg body weight) caused a remarkable induction of hepatic alkaline phosphatase, which increased linearly in the homogenate starting at 5--6 h and reached a maximum level (14-fold of the control activity) at 20--22 h after the drug treatment. In the plasma membrane, however, the increase in specific activity and the recovery of alkaline phosphatase were greatly inhibited up to 12 h after the treatment. Such an inhibitory effect of colchicine was confirmed by a combination experiment of the drug treatment with bile duct ligation; in the plasma membrane elevation of the enzyme induced by bile duct ligation was also greatly retarded by colchicine. The subcellular distribution of the enzyme activity in livers was determined among the four groups of rats with or without bile duct ligation and/or colchicine administration taken at 8 h after each treatment. In the control and the bile duct-ligated livers, the highest specific activity was observed in the plasma membrane fraction, while in the colchicine-treated livers, with or without bile duct ligation, the highest activity was found in the Golgi fractions. These results indicate that the Golgi membranes enriched with the induced enzyme were blocked by the drug to prevent migration toward the plasma membranes enriched with the induced enzyme were blocked by the drug to prevent migration toward the plasma membrane, thus demonstrating involvement of the Golgi complex in the translocation route of newly synthesized alklaine phosphatase to the plasma membrane.

Taxol, a potent promoter of microtubule assembly, inhibits secretion of plasma proteins in cultured rat hepatocytes

Abstract Effects of taxol, a potent promoter of microtubule polymerization, on protein secretion were studied in primary cultured rat hepatocytes. Treatment of cells with 5 × 10 −5 M taxol caused 70% inhibition of secretion as compared with that of the control cells. However, analyses of newly synthesized albumin and α 1 -protease inhibitor demonstrated that the intracellular processing of these proteins was not affected by the drug.

Proteolytic cleavages of proalbumin and complement Pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease☆

We have recently purified and characterized a truncated soluble form of furin from which the predicted transmembrane domain and cytoplasmic tail were deleted (Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) J. Biol. Chem. 267, 16094-16099). Our results showed that furin resembles the yeast Kex2 protease with respect to both its enzymic properties and substrate specificity. Here we demonstrate that the soluble form of furin is capable of converting the precursors of albumin and the third component of complement (proalbumin and pro-C3, respectively) in vitro to mature proteins. Thus furin mimics the Ca(2+)-dependent proalbumin and pro-C3 convertases found in the Golgi membranes (Brennan, S. O., and Peach, R. J. (1988) FEBS Lett. 229, 167-170; Oda, K. (1992) J. Biol. Chem. 267, 17465-17471). Furthermore we show that the variant alpha 1-antitrypsin Pittsburgh, which is a specific inhibitor of the Golgi proalbumin convertase, inhibits not only the Golgi pro-C3 convertase, but also the soluble furin. These results suggest a role for furin in the cleavage of proproteins transported via the constitutive pathway.

Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids

When expressed in COS cells, human prorenin was secreted into the medium without being processed to an active renin. Co-expression of furin, a mammalian homologue of the yeast KEX2 gene product, did not affect proteolytic processing of prorenin. A mutant proreninR-4 constructed by site-directed mutagenesis of Pro (-4) to Arg was not cleaved by an endoprotease in the COS cell. However, proreninR-4 was detectably cleaved to yield the active renin upon co-transfection with furin DNA, indicating that Arg at position -4 is important for recognition and processing by furin in addition to the absolute requirement for paired basic amino acids. Another mutant precursor in which Leu (+1) of proreninR-4 was replaced with Ser was found to be much more efficiently processed than proreninR-4, regardless of co-expression of furin. The results suggest that not only a basic amino acid at position -4 but also Leu at position +1 significantly affect the processing of prorenin catalyzed by the COS cell endoprotease or furin.

Intracellular processing of complement pro-C3 and proalbumin is inhibited by rat α1-protease inhibitor variant (Met352→Arg) in transfected cells

Complement C3, when its cDNA was transfected into COS-1 cells, was synthesized as a precursor, pro-C3, which was intracellularly processed into the alpha and beta subunits, although not completely. A cDNA for rat alpha 1-protease inhibitor (alpha 1-PI) was mutated in vitro to encode its variant with the modified active site (Met352----Arg). In cells co-transfected with the mutant alpha 1-PI cDNA and the C3 cDNA, pro-C3 expressed was secreted without being processed into the subunits. Co-transfection of the mutant alpha 1-PI cDNA and the albumin cDNA also resulted in the inhibition of intracellular conversion of proalbumin into serum-type albumin. No inhibition of the processing of each preform was observed in cells co-transfected with the normal alpha 1-PI cDNA. Taken together, the results indicate that the alpha 1-PI variant (Met352----Arg) expressed inhibits specifically an intracellular enzyme which is involved in the proteolytic processing of both pro-C3 and proalbumin.

Selective processing of proalbumin determined by site-specific mutagenesis

Rat proalbumin is cleaved at the dibasic pair Arg-Arg and converted into a mature form with Glu at the NH2 terminus. In the present study site-directed mutagenesis of the albumin cDNA was designed to generate proalbumin variants in which Glu1 was substituted with various amino acid residues. The expression plasmids constructed were transfected into COS-1 cells, and the intracellular processing of proalbumins expressed was examined by labeling experiments. Substitution of Glu1----Ser allowed the expressed proalbumin to be processed as observed for the wild-type precursor. However, replacement of Glu1 with a hydrophobic residue (Val, Leu or Ile) resulted in no processing of proalbumin, despite retaining the same cleavage signal Arg-Arg as above. The results indicate that the residue at position 1 adjacent to the dibasic pair is also important for recognition by the proalbumin-processing enzyme.