Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.     

Protein export at the ER: loading big collagens into COPII carriers

COPII vesicles mediate the export of secretory cargo from endoplasmic reticulum (ER) exit sites. However, of 60-90 nm diameter COPII vesicles are too small to accommodate secreted molecules such as the collagens. The ER exit site-located proteins TANGO1 and cTAGE5 are required for the transport of collagens and therefore provide a means to understand the export of big cargo and the mechanism of COPII carrier size regulation commensurate with cargo dimensions.     

N-glycosylation Dictates Proper Processing of Organic Anion Transporting Polypeptide 1B1

Organic anion transporting polypeptides (OATPs) have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion (ADME) of various drugs, xenobiotics and toxins. Putative N-glycosylation sites located in the extracellular loops 2 and 5 is considered a common feature of all OATPs and some members have been demonstrated to be glycosylated proteins. However, experimental evidence is still lacking on how such a post-translational modification affect the transport activity of OATPs and which of the putative glycosylation sites are utilized in these transporter proteins. In the present study, we substituted asparagine residues that are possibly involved in N-glycosylation with glutamine residues and identified three glycosylation sites (Asn134, Asn503 and Asn516) within the structure of OATP1B1, an OATP member that is mainly expressed in the human liver. Our results showed that Asn134 and Asn516 are used for glycosylation under normal conditions; however, when Asn134 was mutagenized, an additional asparagine at position 503 is involved in the glycosylation process. Simultaneously replacement of all three asparagines with glutamines led to significantly reduced protein level as well as loss of transport activity. Further studies revealed that glycosylation affected stability of the transporter protein and the unglycosylated mutant was retained within endoplasmic reticulum.     

Degradation of secretory immunoglobulin M in B lymphocytes occurs in a postendoplasmic reticulum compartment and is mediated by a cysteine protease.

In 38C B lymphocytes, membrane IgM is expressed on the surface, whereas secretory IgM (sIgM) is rapidly degraded. Here, we localize this degradation and characterize the proteases involved in this process. Upon treatment with brefeldin A, degradation of sIgM in 38C cells was strongly inhibited, as was secretion from the sIgM-secreting D2 hybridoma. Moreover, the brefeldin A-induced Golgi resorption resulted in galactosylation of sIgM and partial resistance to endoglycosidase H. However, sIgM avoided degradation neither due to modified terminal glycosylation nor as a consequence of the brefeldin A-induced altered milieu of the endoplasmic reticulum. When these modifications were prevented by inhibiting retrograde transport with nocodazole or by abrogating terminal glycosylation with swainsonine, sIgM was still rescued from degradation. The unaffected breakdown in the presence of nocodazole also argued against recycling of sIgM to be degraded in the endoplasmic reticulum. Furthermore, upon removal of brefeldin A, degradation of galactosylated sIgM resumed in 38C cells, as did secretion from D2 cells. These results indicate that functional export of proteins from the endoplasmic reticulum is a prerequisite for sIgM degradation. Biochemical characterization of this novel postendoplasmic reticulum/pre-trans-Golgi proteolytic pathway included application of inhibitors to a broad spectrum of proteases. Among the compounds tested, only calpain inhibitor I exerted strong inhibition. The involvement of cysteine protease(s) in the degradation of sIgM was corroborated by the inhibitory effect of diamide. We conclude that B lymphocytes avoid secretion by active and selective targeting of sIgM to a developmentally regulated postendoplasmic reticulum degradation pathway in which degradation is mediated by a cysteine protease.     

Minimal structural and glycosylation requirements for ST6Gal I activity and trafficking

The influence of N-linked glycosylation on the activity and trafficking of membrane associated and soluble forms of the STtyr isoform of the ST6Gal I has been evaluated. We have demonstrated that the enzyme is glycosylated on Asn 146 and Asn 158 and that glycosylation is not required for the endoplasmic reticulum to Golgi transport of the membrane-associated form of the STtyr isoform. In addition, N-linked glycosylation may stabilize the protein but is not absolutely required for catalytic activity in vivo. In contrast, soluble forms of the protein consisting of amino acids 64-403, 89-403, and 97-403 are efficiently secreted and active in their fully glycosylated forms, but retained in the endoplasmic reticulum and inactive in their unglycosylated forms. These results suggest that membrane associated and soluble forms of the STtyr protein have different requirements for N-linked glycosylation. Elimination of the oligosaccharide attached to Asn 158 in the full length STtyr single and double glycosylation mutants generates proteins that are not cleaved and secreted but stably localized in the Golgi, like the STcys isoform of the ST6Gal I. This stable Golgi localization is correlated with the observation that these two mutants are active in in vivo assays but inactive in in vitro assays of membrane lysates. We predict that removal of N-linked oligosaccharides leads to an increased ability of the STtyr protein to self-associate or oligomerize which subsequently allows more stable retention in the Golgi and increased aggregation and inactivity when membranes are lysed in the in vitro activity assays.     

The N-linked oligosaccharides at the amino terminus of human apoB are important for the assembly and secretion of VLDL

We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N(158)) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N(158), N(956), N(1341), N(1350), and N(1496)), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by >50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d < 1.02 and d > 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB.     

Post-translational modification of bone morphogenetic protein-1 is required for secretion and stability of the protein

Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn(91) (prodomain), Asn(142) (metalloproteinase domain), Asn(332) and Asn(363) (CUB1 domain), Asn(599) (CUB3 domain), and Asn(726) in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn(726) presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.     

ADD66, a gene involved in the endoplasmic reticulum-associated degradation of alpha-1-antitrypsin-Z in yeast, facilitates proteasome activity and assembly

Antitrypsin deficiency is a primary cause of juvenile liver disease, and it arises from expression of the "Z" variant of the alpha-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retrotranslocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed previously, and a gene, ADD66, was identified that facilitates A1PiZ turnover. We report here that ADD66 encodes an approximately 30-kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Delta mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also seems to be a proteasome substrate. Consistent with its role in ER-associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC), and they provide the first link between PAC activity and ERAD.     

Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease

We have examined the topology of the yeast arginine permease, a plasma-membrane protein with multiple membrane-spanning domains. Using fusions of the permease with the glycosylatable secreted yeast protein, acid phosphatase, we identified membrane-spanning sequences that can translocate adjacent acid phosphatase across the membrane of the endoplasmic reticulum (ER), as measured by in vitro glycosylation. Examination for the presence or absence of glycosylation in a systematic series of such fusions gave an internally consistent model for the lumenal or cytoplasmic disposition of the acid phosphatase reporter, defining the topology of the permease. The phenotypes of a further set of arginine permease gene fusions with portions of the gene for the secreted protein, killer toxin, suggest that the pathways of export of membrane and secreted proteins need not be functionally distinct.     

Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.     

Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.     

Elevated synthesis of human alpha 1-antitrypsin hinders the secretion of murine alpha 1-antitrypsin from hepatocytes of transgenic mice

alpha 1-Antitrypsin (AAT) is a major hepatic secretory protein. The elevated synthesis of human AAT within hepatocytes of transgenic mice results in its accumulation within a subset of distended cisternae of the rough endoplasmic reticulum. The protein does not accumulate in large insoluble aggregates as is the case for the human PiZ AAT variant. Furthermore, the accumulated protein is not associated with immunoglobulin heavy chain binding protein. Transgenic animals exhibiting an elevated synthesis and subsequent intrahepatic accumulation of human AAT exhibit reduced serum levels of murine AAT as a result of its hindered secretion and accumulation within the rough endoplasmic reticulum. Interestingly, the secretion of murine transferrin and albumin which represent glycosylated and non-glycosylated hepatic secretory proteins, respectively, is unaffected. Overall, these results demonstrate that the elevated synthesis of human AAT can hinder the export of murine AAT from the hepatic rough endoplasmic reticulum in an apparently specific manner.     

The influence of topology and glycosylation on the fate of heterologous secretory proteins made in Xenopus oocytes

Secretory proteins made in Xenopus laevis oocytes under the direction of heterologous messenger RNA are modified, topologically segregated and exported. Thus the oocyte may serve as a useful surrogate secretory system and we have studied some of the factors governing access to the export pathway. Unglycosylated chicken ovalbumin, synthesized and trapped in the cytosol, is not secreted but glycosylated ovalbumin, found sequestered within vesicles, is exported from oocytes. However, ovalbumin, which is transferred across the endoplasmic reticulum in the presence of tunicamycin and which is indistinguishable by immunoprecipitation, by two-dimensional gel electrophoresis and by concanavalin-A--Sepharose binding from the cytosolic form, is still secreted. Guinea-pig milk proteins and human interferon are also exported from tunicamycin-treated frog cells. These observations demonstrate that access to the endoplasmic reticulum but not glycosylation is a mandatory intermediate step in secretion, and emphasize the advantages of the oocyte as a surrogate system for the study of the later events in the gene expression pathway.     

Intracellular degradation of the transport-impaired human PiZ alpha 1-antitrypsin variant. Biochemical mapping of the degradative event among compartments of the secretory pathway

The naturally occurring PiZ and Pi NullHong Kong variants of the human secretory protein alpha 1-antitrypsin (AAT) are retained within an early compartment of the secretory pathway. Intracellular degradation of these transport-impaired secretory proteins is initiated 30-45 min following their synthesis and translocation into the endoplasmic reticulum (ER). Interestingly, the overall rate of degradation of the retained mutant protein is significantly accelerated when all subcellular compartments are buffered at pH 6. In contrast, degradation is virtually abolished when intravesicular compartments are buffered at pH 8. However, despite this pH sensitivity neither lysosomotrophic amines, leupeptin, or leucine methyl ester have an apparent effect on the intracellular removal of the PiZ variant. The inability of a variety of inhibitors of ER-to-Golgi protein trafficking to hinder the degradative process suggests that degradation of the PiZ variant occurs prior to its delivery to the Golgi complex. To biochemically map the subcellular site of the degradation of the retained mutant protein, a recombinant truncated PiZ variant containing the tetrapeptide KDEL at its carboxyl terminus (a signal for sorting luminal proteins from a post-ER compartment back to the ER) was expressed in cells. Attachment of this ER-recycling signal to the recombinant protein prevented its intracellular degradation. These findings indicate that degradation of the PiZ variant occurs following its export from the ER.     

Cytosolic and nuclear aggregation of the amyloid beta-peptide following its expression in the endoplasmic reticulum

Misfolded secretory and membrane proteins are known to be exported from the endoplasmic reticulum (ER) to the cytosol where they are degraded by proteasomes. When the amount of exported misfolded proteins exceeds the capacity of this degradation mechanism the proteins accumulate in the form of pericentriolar aggregates called aggresomes. Here, we show that the amyloid beta-peptide (Abeta) forms cytosolic aggregates after its export from the ER. These aggregates share several constituents with aggresomes. However, Abeta aggregates are distinct from aggresomes in that they do not accumulate around the centrosome but are distributed randomly around the nucleus. In addition to these cytosolic aggregates, Abeta forms intranuclear aggregates which have as yet not been found for proteins exported from the ER. These findings show that proteins exported from the ER to the cytosol which escape degradation by the proteasome are not necessarily incorporated into aggresomes. We conclude that several distinct aggregation pathways may exist for proteins exported from the ER to the cytosol.     

Tipping the delicate balance: defining how proteasome maturation affects the degradation of a substrate for autophagy and endoplasmic reticulum associated degradation (ERAD)

An increasing body of data links endoplasmic reticulum (ER) function to autophagy. Not surprisingly, then, some aberrant proteins in the ER can be destroyed either via ER associated degradation (ERAD), which is proteasome-mediated, or via autophagy. One such substrate is the "Z" variant of the alpha-1 protease inhibitor (A1Pi), variably known as A1Pi-Z or AT-Z ("anti-trypsin, Z variant"). The wild type protein is primarily synthesized in the liver and is secreted. In contrast, AT-Z, like other ERAD substrates, is retro-translocated from the ER and delivered to the proteasome. However, AT-Z can form high molecular weight polymers that are degraded via autophagy, and cells that accumulate AT-Z polymers ultimately succumb, which leads to liver disease. Therefore, identifying genes that have an impact AT-Z turnover represents an active area of research. To this end, a yeast expression system for AT-Z has proven valuable. For example, a recent study using this system indicates that the activity of a proteasome assembly chaperone (PAC) is critical for maximal AT-Z turnover, which suggests a new role for PACs. Because PACs are conserved, it will be critical to analyze whether these dedicated chaperones are implicated in other diseases associated with ERAD and autophagy.     

Mutations of rat surfactant protein A have distinct effects on its glycosylation,secretion, aggregation and degradation

Aims

Surfactant protein A (SP-A) plays critical roles in the innate immune system and surfactant homeostasis of the lung. Mutations in SP-A2 of the carbohydrate recognition domain (CRD) impair its glycosylation and are associated with pulmonary fibrosis in humans. We aim to examine how mutations in SP-A that impair its glycosylation affect its biological properties and lead to disease.

Main methods

We generated rat SP-A constructs with two types of mutations that impair its glycosylation: N-glycosylation site mutations (N21T, N207S and N21T/N207S) and disease-associated CRD mutations (G231V, F198S). We transfected these constructs into Chinese hamster ovary (CHO)-K1 cells and assessed biochemical differences in cellular and secreted wild-type and mutant SP-As by western blot, immunofluorescence, and sensitivity to enzymatic digestion.

Key findings

Mutations of the CRD completely impaired SP-A secretion, whereas mutations of N-glycosylation sites had little effect. Both types of mutations formed nonidet p-40 (NP-40) insoluble aggregates, but the aggregates only from CRD mutations could be partially rescued by a chemical chaperone, 4-phenylbutyrate acid (4-PBA). The majority of CRD mutant SP-A was retained in the endoplasmic reticulum. Moreover, both types of mutations reduced SP-A stability, with CRD mutant SP-A being more sensitive to chymotrypsin digestion. Both types of soluble mutant SP-A could be degraded by the proteasome pathway, while insoluble aggregates could be additionally degraded by the lysosomal pathway.

Significance

Our data provide evidence that the differential glycosylation of SP-A may play distinct roles in SP-A secretion, aggregation and degradation which may contribute to familial pulmonary fibrosis caused by SP-A2 mutations.     

Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.     

Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N-Terminal Truncation of mGluR1α

The metabotropic glutamate receptor mGluR1alpha in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide-bonded dimer on sodium dodecyl sulphate-polyacrylamide gels. Dimerization of mGluR1alpha takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N-terminal domain of mGluR1alpha, truncated just before the first transmembrane domain (NT-mGluR1alpha), we show that the N-terminal domain is secreted as a soluble disulphide-bonded dimeric protein. In addition, the truncated N-terminal domain can form heterodimers with mGluR1alpha when both proteins are cotransfected into HEK 293 cells. However, mGluR1alpha and its splice variant mGluR1beta did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N-terminal domain of mGluR1alpha is sufficient for dimer formation, other domains in the molecule must regulate the process.     

Soluble aggregates of the human PiZ alpha 1-antitrypsin variant are degraded within the endoplasmic reticulum by a mechanism sensitive to inhibitors of protein synthesis.

Greater than 85% of the transport-impaired PiZ variant of human alpha 1-antitrypsin is retained within transfected mouse hepatoma cells and is subjected to intracellular degradation (Le, A., Graham, K., and Sifers, R.N. (1990) J. Biol. Chem. 265, 14001-14007). The retained protein undergoes a discrete size reduction that results from the modification of its endoglycosidase H-sensitive oligosaccharides and is inhibited by 1-deoxymannojirimycin. Metabolic poisons and inhibitors of protein synthesis perturb the intracellular degradation of the retained protein but do not affect its size reduction. The ability of metabolic poisons to influence the degradation of the PiZ variant in cells treated with brefeldin A indicates that export of the macromolecule from the endoplasmic reticulum (ER) is not the energy-dependent component of its degradation. Subcellular fractionation experiments have verified that both the size reduction and degradation of the retained PiZ variant occur within the rough ER. Finally, sedimentation velocity centrifugation analysis of radiolabeled cell extracts has indicated that approximately 80% of the PiZ variant consists as soluble aggregates immediately after its synthesis. An inability to detect more extensive aggregation during the retention period supports our previous conclusion that only a small fraction of the macromolecules actually form large insoluble aggregates (Graham, K.S., Le, A., and Sifers, R.N. (1990) J. Biol. Chem. 265, 20463-20468). Overall, these findings indicate that soluble aggregates of the PiZ variant are degraded within the ER by a mechanism sensitive to inhibitors of protein synthesis.