Novel human proalbumin variant with intact dibasic sequence facilitates identification of its converting enzyme

We describe here the identification of a new genetic variant of human proalbumin with an N-terminal sequence of Arg-Gly-Val-Phe-Arg-Arg-Val-Ala-His-Lys-. Proalbumin Blenheim (10%) and mature albumin Blenheim (38%) with an initial sequence of Val-Ala-His-Lys-make up nearly half the serum albumin in affected individuals. Despite retaining an intact dibasic processing site, proalbumin Blenheim (1 Asp----Val) enters the circulation unprocessed. The observed ratio of proalbumin to albumin can be accounted for by proteolysis in the periphery. Employed as a potential substrate, proalbumin Blenheim provides a unique means of identifying the physiologically relevant proalbumin convertase. In vitro studies showed that the variant is readily cleaved by trypsin. However, it is not cleaved by the proposed proalbumin convertase, a membrane-bound Ca2+-dependent proteinase prepared from rat liver Golgi vesicles, which gives authentic cleavage of normal human proalbumin.     

Ligand-binding properties of proalbumin Christchurch.

Proalbumin Christchurch, a circulating variant of human serum albumin, is secreted from the liver without cleavage of the hexapeptide situated at the N-terminal end of the peptide chain of proalbumin. We compared ligand-binding properties of proalbumin Christchurch and of normal albumin A from the same individual in order to test the effect of the presence of the hexapeptide. The two albumin forms exhibited similar affinities for palmitate, bilirubin, 8-anilinonaphthalene-1-sulphonate and Bromocresol Green. The patterns of endogenous fatty acids bound to the two forms of albumin were slightly different, although the differences were probably not of physiological significance. From these studies it would appear that the propeptide of proalbumin does not alter the protein conformation in such a way as to alter binding sites for organic anions.     

Structure and properties of albumin Tokushima and its proteolytic processing by cathepsin B in vitro

Albumin Tokushima is a Japanese genetic variant of human serum albumin. Two homozygous and 6 heterozygous subjects with this variant were found in a family. Albumin Tokushima was purified from sera of the homozygous subjects. Its amino acid composition and amino-terminal sequence were determined and compared with those of a normal serum albumin. Albumin Tokushima with the amino-terminal sequence of Arg-Gly-Val-Phe-His-Arg-Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys- Asp- Leu-Gly-Glu-Glu-Asn-Phe was found to be the same abnormal proalbumin as proalbumin Lille (Abdo, Y. et al. (1981) FEBS Lett. 131, 286-288). The isoelectric points of albumin Tokushima were pH 4.70 and 4.90 as compared with pH 5.05 and 5.25 of a normal serum albumin. Albumin Tokushima was converted to normal serum albumin by purified cathepsin B in vitro. Albumin Tokushima can bind Ni2+ at 4 degrees C but binds little at 37 degrees C.     

Evidence for the coupling of biosynthesis and secretion of serum albumin in the rat. The effect of colchicine on albumin production

1. By using isotopic-dilution techniques it was found that colchicine causes a slight increase in the proalbumin content of liver, from 0.63+/-0.06 to 0.83+/-0.10mg/g of liver, but has no effect on albumin content (0.50+/-0.05mg/g of liver). All the proalbumin and 67% of the albumin is found in vesicles from which they are liberated by detergents. 2. Colchicine inhibits secretion of albumin, decreases the rate of conversion of proalbumin into albumin and decreases the rate of incorporation of l-[1-(14)C]leucine into proalbumin. 3. Balance studies in vivo show that all the (14)C appearing in serum albumin can be accounted for by the flow of (14)C through the proalbumin, in the presence or absence of colchicine. 4. When cycloheximide is given to the rats, 2min after [(14)C]leucine, further synthesis of protein stops. The label in proalbumin disappears and the proalbumin content of the liver falls, so as to account for the albumin appearing in the plasma. This occurs both in the presence and in the absence of colchicine. By contrast, there is little change in liver albumin. Studies with isolated perfused livers are in agreement with the above. Lumicolchicine has no effect on any of these systems at doses at which colchicine exerts its action. 5. These results suggest that biosynthesis and conversion of proalbumin into albumin, and secretion of serum albumin are controlled at each step.     

Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin

Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp314-->Val (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg-2-->Cys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: GAT-->GTT (albumin Brest) and CGT-->TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg-6. In addition, part of normal albumin had lost Asp1. Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding.     

In vivo effect of colchicine on hepatic protein synthesis and on the conversion of proalbumin to serum albumin

Treatment of rats with 0.5-25 mumol/100 g body weight of colchicine for 1 h or more caused an inhibition of hepatic protein synthesis. This effect was not seen if animals were exposed to colchicine for less than 1 h. The delayed inhibition of protein synthesis affected both secretory and nonsecretory proteins. Treatment with colchicine (15 mumol/100 g) for 1 h or more caused the RNA content of membrane-bound polysomes to fall but did not change the polysomal profile of this fraction. By contrast, the total RNA content in the free polysome cell fraction was increased, and this was due to the presence of more ribosomal monomers and dimers. Electron microscope examination of the livers from rats treated for 3 h with colchicine showed an accumulation of secretory vesicles within the hepatocytes and a general distention of the endoplasmic reticulum. Administration of radioactive L-leucine to the rats led to an incorporation of radioactivity into two forms of intracellular albumin which were precipitable with antiserum to rat serum albumin but which were separable by diethylaminoethyl-cellulose chromatography. One form has arginine at the amino-terminal position and is proalbumin, and the other form, which more closely resembles serum albumin chromatographically, has glutamic acid at its amino terminus. Only proalbumin was found in rough and smooth endoplasmic reticulum fractions and in a Golgi cell fraction wich corresponds morphologically to mostly empty and partially filled secretory vesicles. However, in other Golgi cell fractions which were filled with secretory products, both radioactive proalbumin and serum albumin were found. This indicates that proalbumin is converted to serum albumin in these secretory vesicles just before exocytosis. Colchicine delayed the discharge of radioactive albumin from these filled secretory vesicles and caused an accumulation of both proalbumin and serum albumin within these cell fractions.     

Analytical cation-exchange chromatography to assess the identity,purity, and N-terminal integrity of human lactoferrin

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants.     

Weakly basic amines inhibit the proteolytic conversion of proalbumin to serum albumin in cultured rat hepatocytes

Effects of weak amines on the proteolytic conversion of proalbumin to serum albumin were studied in primary culture of rat hepatocytes. In control culture proalbumin was converted to serum albumin before discharge into the medium. However, in the presence of chloroquine the conversion to serum albumin was inhibited and proalbumin per se was released into medium. A similar inhibition of the processing was also observed in the presence of other amines such as methylamine and NH4Cl. Thus weak amines mimic the carboxylic ionophore monensin with regard to the effect on proalbumin conversion [Oda & Ikehara (1982) Biochem. Biophys. Res. Commun. 105, 766-772]. Since proteolytic conversion of proalbumin is believed to occur at the Golgi complex, these results suggest that weakly basic amines perturb the Golgi complex in addition to lysosomes and endosomes.     

Proalbumin is processed to serum albumin in COS-1 cells transfected with cDNA for rat albumin

Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells.     

Secretion of proalbumin by canavanine-treated Hep-G2 cells

The two processing sites in the conversion of preproalbumin to albumin are marked by arginine residues. Therefore, to study the mechanisms of albumin processing and secretion, the arginine residues of nascent albumin were replaced with canavanine by the incubation of Hep-G2 cells with this arginine analog. During a 4-h interval, canavanine inhibited (67%) the secretion of nascent albumin and increased the intracellular transit time of albumin secretion from 24 to 39 min. At 1 h, canavanine inhibited total protein synthesis by 19% and albumin synthesis by about 40%. Both the intracellular and secreted albumin produced by canavanine-treated cells were analyzed by DEAE-cellulose chromatography and were found to be more acidic than normal proalbumin and albumin. Further analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the albumin produced and secreted by canavanine-treated cells appeared to have a larger molecular weight (by 4000) than serum albumin. The canavanine-treated cells were incubated with L-[3H]leucine and L-[3H]phenylalanine and the location of radioactive L-leucine and L-phenylalanine in the 30 NH2-terminal amino acid residues of secreted albumin was determined. The results indicated that canavanine-treated cells secreted proalbumin (79%) and also some fully processed albumin (21%). Preproalbumin was not secreted. Untreated Hep-G2 cells mostly secreted fully processed serum albumin (93%) with only traces of proalbumin (7%).     

Conversion of proalbumin into serum albumin in the secretory vesicles of rat liver

The conversion site of proalbumin into serum albumin was investigated in the subcellular fractions of rat liver labeled with [³H] leucine in vivo. In the cisternae-rich fraction of the Golgi complex as well as in the microsomal fraction most of the labeled albumin was detected as proalbumin, while in the secretory vesicles, which were obtained in increased amount by oral administration of ethanol, more than 70% of the labeled albumin was found as serum type, indicating that conversion of proalbumin into serum albumin occurs within the secretory vesicles in rat liver. Little accumulation of albumin was observed in colchicine-treated rats.     

Bovine microsomal albumin: Amino terminal sequence of bovine proalbumin

Bovine liver microsomes contain an albumin having an apparent isoelectric point approximately 0.3 pH unit in excess of bovine serum albumin. Sequence analysis of the purified protein shows that the first ten residues at the amino terminus are: $\text{Arg-Gly-Val-Phe-Arg-Arg}$-Asp-Thr-His-Lys. The data suggest that the hexapeptide (underlined), identical to that found in proalbumin from rat liver, is attached to the amino terminus of bovine serum albumin (the last four residues). By analogy with the rat liver system, this protein therefore is bovine proalbumin, a precursor of bovine serum albumin.     

Intracellular processing of complement pro-C3 and proalbumin is inhibited by rat alpha 1-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.     

Circulating proalbumin associated with a variant proteinase inhibitor

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1–5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, α₁-antitrypsin Pittsburgh (358Met→Arg) [2]. The infeerred structure of human proalbumin was confirmed as ArgGlyValPheArgArgAlb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.     

Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

Proalbumin to albumin conversion by a proinsulin processing endopeptidase of insulin secretory granules

A lysate of purified insulin secretory granules, which contains two types of proinsulin processing activity (type 1, Arg-Arg-directed and type II, Lys-Arg-directed (Davidson, H.W., Rhodes, C.J., and Hutton, J. C. (1988) Nature 333, 93-96), was found to process proalbumin by specific proteolytic cleavage of the COOH-terminal side of the Arg-2-Arg-1 sequence. The subcellular distribution of proalbumin processing activity in insulinoma tissue paralleled that for proinsulin conversion and occurred principally in a secretory granule fraction. Cleavage appeared to result from the Arg-Arg-directed type 1 proinsulin processing endo-peptidase. It was Ca2+-dependent (K0.5 activation = 1.0-1.5 mM Ca2+), unaffected by group-specific inhibitors of serine, cysteinyl, or aspartyl proteinases, and had an acidic pH optimum (5.5). Active-site inhibitor studies showed this activity had a preference for dibasic over monobasic amino acid sequences and indicated that the sequence of the dibasic site was an important determinant of the susceptibility of the substrate to cleavage. The activity did not process the proalbumin Christchurch mutant (Arg-2-Arg-1 to Arg-2-Gln-1). It was inhibited by the variant alpha 1-antitrypsin Pittsburgh (Met358 to Arg358; K0.5 = 100 nM) but not by other related proteins normally co-secreted with albumin from hepatocytes, namely alpha 1-antitrypsin M, alpha 2-macroglobulin, or antithrombin III. The insulin secretory granule proalbumin processing activity was indistinguishable from a proalbumin endopeptidase reported in rat liver membranes and similar to the yeast KEX-2 protease. These findings suggest that a highly conserved set of proprotein endopeptidases exists, which are specific for a dibasic sequence but broadly specific for proprotein substrates. Such enzymic activities appear to be active within both the constitutive and regulated pathways of secretion. Intraorganellar Ca2+ and pH appear to play a key role in regulating their activities.     

Putative convertase involved in the proteolytic conversion of rat proalbumin to serum albumin

We have found a proteolytic activity in Golgi membranes which efficiently converts [35S]methionine-labeled proalbumin, isolated from pulse-labeled rat hepatocytes in culture, to serum albumin in an in vitro assay system. The proalbumin-converting activity was dependent on Ca2+ and the maximum activity was observed at pH 5.5-6.0. Since the enzyme activity was found to be resistant not only to both leupeptin and E-64 but also to thiol-blocking reagents, it is unlikely that cathepsin B is involved in the proteolytic conversion of proalbumin occurring in the Golgi complex.     

Calcium-dependent Golgi-vesicle fusion and cathepsin B in the conversion of proalbumin into albumin in rat liver.

1. An enzyme from rat liver that converts proalbumin into albumin is described. Partial purification, inhibitor studies and the conditions for maximum activity suggest that the enzyme is cathepsin B. 2. A membrane-bound enzyme, located mainly in lysosomes, also converts proalbumin into albumin. This appears to be a membrane-bound form of cathepsin B. 3. Isolated Golgi vesicles, incubated under conditions suitable for cathepsin B, convert endogenous proalbumin into albumin. 4. This conversion in Golgi vesicles has an absolute requirement for Ca2+ at micromolar concentrations. Mg2+ does not affect or substitute for Ca2+. Both the proalbumin and the albumin formed from it are intravesicular. 5. Converting activity is enhanced by pretreatment with the known chemical fusogen, poly(ethyleneglycol). 6. Vesicles preincubated at pH above 7 in the presence of dithiothreitol show a marked fall in converting activity. This can be partially restored by incubation with native vesicles. These results suggest that vesicle fusion is a requirement for conversion of proalbumin into albumin.     

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.     

Human albumin genetic variants: an attempt at a classification of European allotypes

The relative mobility of albumin and proalbumin genetic variants was estimated by means of cellulose acetate electrophoresis performed with three buffer systems at different pH (8.6, 5.0, and 6.9) after addition of a reference protein and dilution of sera. Numerous experiments using samples of reference variants corroborated the accuracy and reproducibility of this technique. The estimation of the variants' relative mobility at three pH allowed us to distinguish three fast-moving variants (Gent, Vanves, and Reading) and five slow-moving variants (Sondrio, Roma, Christchurch, Lille, and B) in the French population. The frequency of alloalbuminemia in this population is .0004 and is characterized by the high occurrence of albumin B and of the two proalbumin variants, Christchurch and Lille. In order to classify the variants of European origin, the methodology that we developed, owing to its more resolutive possibilities, should be employed as a first step in their identification until establishment of a structural nomenclature making mention of the amino acid substitution characterizing each variant.