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 precursor (pro-C3), which was processed to the mature form with alpha- and beta-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.     

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.     

Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases.

The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.     

Properties, biosynthesis and processing of a sulfur-rich protein in Brazil nut (Bertholletia excelsa H.B.K.)

An abundant seed protein, which is exceptionally rich in the sulfur-containing amino acids, methionine (18%) and cysteine (8%), is synthesized in Brazil nut embryos about 9 months after flowering. This sulfur-rich protein consists of two low-molecular-mass polypeptide components, a 9-kDa polypeptide and a 3-kDa polypeptide. The two-subunit polypeptides associate through disulfide linkage(s) to form a 12-kDa protein molecule. We have demonstrated through in vitro translation studies, using RNA from 9-month-old embryos, that the sulfur-rich protein is synthesized as a larger precursor polypeptide of 18 kDa. In addition, data from in vivo labelling studies of 9-month-old Brazil nuts suggest that there are two intermediate precursors of the sulfur-rich protein, one of 15 kDa and another of 12 kDa. One of these precursors, the 12-kDa polypeptide, accumulates for a 2-month period in the developing embryos. From these data we infer that at least three stepwise cleavages are involved in the maturation of the sulfur-rich protein from its 18-kDa precursor.     

Intracellular and secreted proteins of density-inhibited, serum-arrested and proliferating mouse embryo cells

Short-term labelling of secondary cultures of mouse embryo fibroblasts with [14-C] aminoacids enabled the identification and quantitation of proteins specific for quiescent and proliferative stages. Intracellular and secreted proteins of cells maintained under different growth conditions were resolved in high resolution SDS-polyacrylamide gradient gels. Two proteins, identified as fibronectin and procollagens and a 34 000 D polypeptide were found to be secreted by all three types (density-arrested, serum arrested and proliferating) of cells. Both types of arrested cells exclusively secreted a 375 000 D protein while the proliferating cells specifically secreted a 48 000 D polypeptide. During progression of cells from quiescence to proliferation, two intracellular proteins showed major variations. A 205 000 D intracellular protein was found to be synthesized in higher amounts by proliferating cells than by arrested cells. Another protein, identified as actin, showed a marked increase in synthesis following the release of cells from serum arrest. The arrested cells showed reduced levels of actin synthesis and the turning-off process in the synthesis of actin was found to be relatively slow as the cells entered into quiescence.     

Functional studies on the secreted form of human C4 (C4s), two incompletely processed two-subunit C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and pro-C4

The functional properties of the secreted form of C4 (C4s), which has a Mr approximately 5000 greater than the predominant C4 molecule found in plasma (C4p), two incompletely processed two-chain C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and the extracellular C4 precursor (designated pro-C4(E)] were evaluated. All four molecules are secreted in parallel by a human hepatoma-derived cell line (Hep G2). Secretion of hemolytically active C4 is linear up to approximately 12 hr, peaks at 24 hr, and then progressively decreases over the next 48 hr. This loss of C4s functional activity parallels the proteolytic conversion of C4s to C4bs. To compare the hemolytic efficiencies of C4s and C4p, a solid-phase competitive radioimmunoassay was developed to permit measurement of the small quantities of C4 antigen in these cultures. The hemolytic efficiencies of C4s and C4p were similar. These results indicate that extracellular processing of C4s to C4p does not modulate the hemolytic activity of the molecule. Consistent with their ability to bind methylamine, both the alpha s-chain and the alpha - gamma subunit undergo denaturation-induced autolysis. The extracellular and intracellular pro-C4 molecules are also sensitive to autolytic cleavage. Interestingly, the beta - alpha subunit is resistant to autolysis. In experiments in which C4s and C4p were cleaved by C1-s to C4bs, C4(beta - alpha + gamma), C4(beta + alpha - gamma), and pro-C4(E) were resistant to C1-s cleavage and thus hemolytically inactive relative to C4s. These data indicate that processing of C4 to a three-chain structure is required to provide the proper conformation for efficient activation by C1.     

A novel proteolytic activity in serum processes rat prohaptoglobin

The heterotetrameric plasma glycoprotein rat haptoglobin previously was shown to be synthesized by hepatocytes in a precursor form, prohaptoglobin, which contains one alpha-subunit region and one beta-subunit region. Two of these molecules, each with a molecular weight of 45,000, are joined by a disulfide bond and subsequently the subunit regions of each polypeptide are separated by site-specific proteolysis, yielding the tetrameric native protein. Although some of this processing occurs intracellularly, a substantial proportion of the prohaptoglobin is secreted [J. M. Hanley, T. H. Haugen, and E. C. Heath (1983) J. Biol. Chem. 258, 7858-7869]. However, a proteolytic activity was found in rat plasma and serum which also is capable of site-specific cleavage of prohaptoglobin. Further investigation of this novel activity has demonstrated that it cleaves prohaptoglobin accurately, in the same site-specific manner as the intracellular protease, and that it most likely is not a serine protease or a metalloenzyme but can be inhibited by sulfhydryl-reactive compounds. Furthermore, it appears to be synthesized and secreted by hepatocytes, and thus may be identical to the intracellular processing protease.     

Complete nucleotide and derived amino acid sequences of sex-limited protein (Slp), nonfunctional isotype of the fourth component of mouse complement (C4)

The nucleotide sequence coding for sex-limited protein (Slp), the testosterone-regulated isotype of the fourth component of mouse complement (C4), has been determined from cloned genomic DNA and cDNA fragments. The complete deduced amino acid sequence for the single chain precursor protein of Slp (pro-Slp) consists of 1716 residues. The mature beta, alpha, and gamma subunits contain 654, 763, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted from the beta-chain, five for the alpha-chain, and none for the gamma-chain. From the comparison with the mouse C4 sequences, an extensive overall sequence homology, 96.0% in nucleotides and 94.2% in amino acids, is observed. Only one deletion/insertion event is recognized between C4 and Slp sequences: three residues near the Cls cleavage site are deleted from Slp. The distribution of cysteine residues is completely conserved between pro-Slp and pro-C4.     

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.     

Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein.

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].     

Characterization of a temperature-sensitive, hexon transport mutant of type 5 adenovirus.

Infection of KB cells at 39.5 degrees C with H5ts147, a temperature-sensitive (ts) mutant of type 5 adenovirus, resulted in the cytoplasmic accumulation of hexon antigen; all other virion proteins measured, however, were normally transported into the nucleus. Immunofluorescence techniques were used to study the intracellular location of viral proteins. Genetic studies revealed that H5ts147 was the single member of a nonoverlapping complementation group and occupied a unique locus on the adenovirus genetic map, distinct from mutants that failed to produce immunologically reactive hexons at 39.5 degrees C ("hexon-minus" mutants). Sedimentation studies of extracts of H5ts147-infected cells cultured and labeled at 39.5 degrees C revealed the production of 12S hexon capsomers (the native, trimeric structures), which were immunoprecipitable to the same extent as hexons synthesized in wild type (WT)-infected cells. In contrast, only 3.4S polypeptide chains were found in extracts of cells infected with the class of mutants unable to produce immunologically reactive hexon protein at 39.5 degrees C. Hexons synthesized in H5ts147-infected cells at 39.5 degrees C were capable of being assembled into virions, to the same extent as hexons synthesized in WT-infected cells, when the temperature was shifted down to the permissive temperature, 32 degrees C. Infectious virus production was initiated within 2 to 6 h after shift-down to 32 degrees C; de novo protein synthesis was required to allow this increase in viral titer. If ts147-infected cells were shifted up to 39.5 degrees C late in the viral multiplication cycle, viral production was arrested within 1 to 2 h. The kinetics of shutoff was similar to that of a WT-infected culture treated with cycloheximide at the time of shift-up. The P-VI nonvirion polypeptide, the precursor to virion protein VI, was unstable at 39.5 degrees C, whereas the hexon polypeptide was not degraded during the chase. It appears that there is a structural requirement for the transport of hexons into the nucleus more stringent than the acquisition of immunological reactivity and folding into the 12S form.     

The primary structure of the fourth component of human complement (C4)-C-terminal peptides

C-terminal CNBr peptides of the three polypeptide chains of C4 were obtained and sequenced. These results supplement previously obtained data, notably the protein sequence derived from cDNA sequencing of pro-C4 (Belt KT, Carroll MC & Porter RR (1984) Cell 36, 907-914) and the N-terminal sequences of the three polypeptides (Gigli I, von Zabern I & Porter RR (1977) Biochem. J. 165, 439-446), to define the complete primary structure of the plasma form of C4. The beta (656 residues), alpha (748 residues), and gamma (291 residues) chains are found in positions 1-656, 661-1408, and 1435-1725 in the pro-C4 molecule.     

Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.     

Complement proteins C2, C4 and factor B. Effect of glycosylation on their secretion and catabolism.

Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.     

Post-translational modification of the fourth component of complement. Effect of tunicamycin and amino acid analogs on the formation of the internal thiol ester and disulfide bonds

The appearance of a functional thiol ester within murine pro-C4 (the intracellular precursor of C4) has been studied. This was assessed by testing the ability of pro-C4 molecules to undergo denaturation-dependent autolytic cleavage. In pulse-chase experiments, [35S]methionine-labeled pro-C4 does not autolyze until approximately 20 min after synthesis by peritoneal macrophages. When intact (not autolyzed) pro-C4 was examined by nonreducing gel electrophoresis, an increase in its apparent Mr was seen, with a time course similar to that for autolysis. Both the capacity to undergo autolytic cleavage and the Mr increase were inhibited by cell culture in the presence of the antibiotic tunicamycin or the threonine analog beta-hydroxynorvaline, both of which inhibit glycosylation. Upon isolation from tunicamycin- or hydroxynorvaline-treated cells, pro-C4 associates with other cell constituents, probably via disulfide bonds. This phenomenon is not seen with the mature (high Mr) form of pro-C4 in control cultures, and can be prevented if the cells are lysed in the presence of a sulfhydryl reagent such as iodoacetamide. These data suggest that the post-translational modification of pro-C4 includes the acquisition of a disulfide-stabilized conformation with a greater apparent Mr. This conformation, along with an intact thiol ester, is necessary for autolytic cleavage to occur.     

Purification and characterization of a single chain precursor C3-protein (pro-C3) from normal human plasma.

Fresh human plasma was treated with proteinase inhibitors and passed through an immunoadsorbent column of Sepharose anti-C3 globulin. The insolubilized C3 was eluted with 5 M guanidine and, after extensive dialysis, was reduced with 0.2 M 2-mercaptoethanol and electrophoresed on SDS-polyacrylamide gels. The bulk of the eluted C3 dissociated into two major protein bands, m.w. 125,000 and 75,000 corresponding to the alpha- and beta-chains of C3. In addition, a nonreducible single polypeptide chain (SPC) with a m.w. value of 197,000 +/- 2,000 similar to the apparent m.w. of unreduced C3 was consistently present. SPC has been purified by elution from SDS (SPC) and found to remain a single polypeptide chain upon re-electrophoresis on SDS gels in the presence of 0.2 M 2-mercaptoethanol. The purified SPC reacted with antisera to denatured C3, C3alpha, and C3beta chains. Additionally, antisera to SPC, also reacted with denatured C3, C3alpha-, and C3beta-chains, revealed a reaction identity between SPC and C3, and detected partial identity between SPC and C3alpha- as well as C3beta-chains. This suggested that SPC and C3 are antigenically related. The amino acid compositions and tryptic peptide maps of SPC and C3 were similar. Based on these findings, it is suggested that SPC must represent a single-chain precursor C3 (pro-C3) in plasma that escaped post-synthetic proteolytic cleavage into a two-subunit chain C3 molecule.     

Molecular analysis of human complement component C5: localization of the structural gene to chromosome 9

A human C5 clone (pC5HG2) was isolated from a cDNA library constructed from Hep G2 mRNA. The DNA sequence showed that the pC5HG2 insert was comprised of 3309 base pairs of pro-C5 coding sequence and 404 base pairs of 3'-untranslated sequence. The derived amino acid sequence contained the entire coding sequence of the C5 alpha-chain, the beta-alpha-chain junction region, and 100 amino acids (approximately 50%) of the beta-chain. Protein sequences of four C5 tryptic peptides were aligned exactly to this sequence and demonstrated that C5 synthesized and secreted by Hep G2 cells is probably identical with plasma-derived C5. Coding sequence alignment of the human C5 sequences with those of murine C5 indicated that 80% of the nucleotides and 79% of the amino acids were placed identically in the two species. Amino acid sequence alignment of the homologous family members C3, C4, and alpha 2-macroglobulin with that of C5 demonstrated 27%, 25%, and 19% identity, respectively. As was found in murine C5, the corresponding thiol ester region of human C5 contained several conserved amino acids, but the critical cysteine and glutamine residues which give rise to the intramolecular thiol ester bond in C3, C4, and alpha 2-macroglobulin were absent in C5, having been replaced by serine and alanine, respectively. With the use of a panel of hamster-human somatic cell hybrids, the C5 gene was mapped to human chromosome 9. In situ chromosomal hybridization studies employing metaphase cells further localized the gene to bands 9q32-34, with the largest cluster of grains at 9q34.1.     

Glycoprotein biosynthesis in myeloma cells. Characterization on nonglycosylated immunoglobulin light chain secreted in presence of 2-deoxy-D-glucose.

The biosynthesis and secretion of a glycosylated, K-type immunoglobulin light chain (K-46) was studied in a mouse myeloma tumor, mineral oil plasmacytoma-46B. Viable single cell suspensions were prepared from excised tumors and optimal conditions were established for incorporation of amino acid and carbohydrate precursors into the protein synthesized and secreted by the cells. The glucose analog, 2-deoxy-D-glucose, was utilized as an inhibitor of glycosylation to determine the role of glycosylation in the biosynthesis, intracellular transport, and export of the protein from the cell. It was determined that 6 mM 2-deoxyglucose prevents the incorporation of glucosamine, mannose, and galactose into secreted protein, but permits the incorporation of leucine at approximately 40% of control values. The nonglycosylated protein, secreted in the presence of 2-deoxyglucose, was characterized as a nonglycosylated form of K-46 light chain by the following criteria: (a) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, (b) reactivity of the nonglycosylated protein with antisera prepared against native, fully glycosylated, K-46 light chain, (c) analysis of the protein by gel filtration techniques, (d) behavior of the protein on lectin-derivatized Sepharose, and (e) analysis of tryptic peptides derived from the protein. We have concluded that 2-deoxyglucose-inhibited cells synthesize and secrete the normal polypeptide chain of K-46 devoid of its carbohydrate side chain indicating that glycosylation is not an essential step in the biosynthesis, intracellular transport, or export of this protein that is normally synthesized and secreted in a glycosylated form. Under conditions of 2-deoxyglucose inhibition, the nonglycosylated form of K-46 light chain constitutes a significantly greater proportion of accumulated intracellular protein, suggesting that the biosynthesis of the polypeptide chain of K-46 light chain proceeds at a nearly normal rate, but that the absence of the carbohydrate side chain of the protein retards, but does not prevent, the intracellular transport of the protein and its export from the tumor cell.     

Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes

Hepatic lipase, a glycoprotein synthesized and secreted by the hepatocyte, binds to sinusoidal endothelium where it is involved in metabolism of lipoprotein phospholipid and triglyceride. To better understand the regulation of hepatic lipase, we investigated the synthesis, post-translational processing, and secretion of the enzyme by isolated rat hepatocytes. Metabolically labeled [35S]methionine hepatic lipase protein, produced by the collagenase-dispersed hepatocytes, was immunoisolated from detergent-solubilized cells and incubation medium at designated times, using a polyclonal rabbit anti-rat hepatic lipase antibody raised against hepatic lipase purified to homogeneity from rat liver post-heparin perfusates. Following polyacrylamide gel electrophoresis and fluorography, radiolabeled hepatic lipase was quantitated by densitometry. Newly synthesized hepatic lipase was rapidly secreted and accumulated in the medium as a 59,000-dalton protein in a manner consistent with a constitutive process. An intracellular 53,000-dalton precursor of the mature 59,000-dalton hepatic lipase was identified by immunoprecipitation. The 53,000-dalton form could also be generated by endoglycosidase digestion of the secreted 59,000-dalton protein. In pulse-chase experiments, the 53,000-dalton protein was converted into the 59,000-dalton form. A 47,000-dalton form of hepatic lipase was immunoisolated from cell lysates only after tunicamycin treatment and could be generated from the secreted 59,000-dalton enzyme by prolonged endoglycosidase digestion. These data show that hepatic lipase is synthesized and rapidly secreted by isolated rat hepatocytes. Further, an intracellular 47,000-dalton precursor peptide can be identified after tunicamycin treatment, which may represent the hepatic lipase polypeptide, presumably after removal of its signal sequence; a 53,000-dalton partially glycosylated peptide exists as a major precursor form in the cell; and the mature 59,000-dalton hepatic lipase is present in the hepatocyte, but it is rapidly secreted.     

Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.