Dissecting glycoprotein quality control in the secretory pathway.

In the early secretory pathway, asparagine-linked glycosylation facilitates the conformational maturation of diverse polypeptides by promoting their physical engagement with the glycoprotein-folding machinery. Misfolded glycoproteins are selectively eliminated from the endoplasmic reticulum by a stringent process of conformation-based quality control. Recent studies indicate that a small ensemble of oligosaccharide-processing enzymes and lectins use the asparagine-linked appendage to orchestrate the selective disposal of numerous transport-defective glycoproteins from the early secretory pathway. The glycan-based disposal system functions as an evolutionarily conserved terminal checkpoint in eukaryote genome expression. That the mechanisms by which glycoprotein substrates are recruited for degradation diverge at the level of signal recognition reflects a previously unappreciated component of cellular differentiation in higher eukaryotes.

Multiple tissues express alpha 1-antitrypsin in transgenic mice and man.

Hepatocytes are considered to be the predominant source of alpha 1-antitrypsin (AAT), the major antiprotease in human plasma. The development of emphysema in the hereditary PiZ AAT deficiency state suggests that inhibition of leukocyte elastase in the lung is a major function of this protein. In addition, patients with AAT deficiency are at increased risk for developing cholestasis in infancy and chronic liver disease as adults. The mechanism for hepatic cell injury, however, is not understood. Transgenic mice that express the normal human AAT gene demonstrate abundant AAT in hepatocytes and specific cell types of numerous nonhepatic tissues. Immunoperoxidase techniques have previously disclosed AAT in many of the cell types seen in transgenic mice; however, the issue of local synthesis vs. endocytosis in these cell types has remained unresolved. In this study, AAT mRNA was seen in a variety of tissues in the transgenic mouse. Immunoelectron microscopy of renal tubular and small intestinal epithelial cells in the transgenic mice demonstrated AAT within the cisternae of the rough endoplasmic reticulum, as in hepatocytes. These findings support the possibility of local synthesis in the various cell types. The results suggest that in addition to maintaining tissue integrity in the lung, the protease/antiprotease balance may have physiological functions in other organs as well.

Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice.

Circulating alpha 1-antitrypsin is synthesized primarily in the liver and secreted into the bloodstream, where it serves as the major protease inhibitor. The PiZ variant of alpha 1-antitrypsin is associated with decreased levels of the protein in sera as a result of its retention within hepatocytes. Homozygosity for the variant allele predisposes individuals to the development of pulmonary emphysema and an increased risk for liver disease. We and others have previously demonstrated that the normal PiM human alpha 1-antitrypsin gene can be properly expressed in the livers of transgenic mice. The PiZ variant of the human alpha 1-antitrypsin gene was introduced into the germline of mice to determine whether the mutant protein would accumulate in mouse hepatocytes and if such accumulation would result in the development of liver damage in an animal model. As expected, the mutant human protein was abundantly synthesized in the livers of the transgenic animals and accumulated within the rough endoplasmic reticulum of hepatocytes as it does in human patients. PiZ mice developed significantly more liver necrosis and inflammation than PiM transgenic mice or control littermates. The degree of liver damage was correlated with the amount of PiZ alpha 1-antitrypsin accumulated in the liver of the different pedigrees of mice. Although 40% of PiZ mice tested were seropositive for mouse hepatitis virus (MHV), the degree of liver damage was not influenced by the MHV seropositivity; rather, it was related only to the presence of accumulated PiZ protein.

Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry's disease.

A simple and sensitive fluorometric method has been described for the differential determination of the activity of lysosomal alpha-galactosidase A and alpha-galactosidase B. The procedure employs 4-methylumbelliferyl-alpha-D-galactopyranoside as substrate and N-acetylgalactosamine as an inhibitor of alpha-galactosidase B, but not of alpha-galactosidase A to differentiate the two activities. This method was shown to be applicable in the differentiation of the two enzyme activities in human tissues and in the diagnosis of the heterozygous and hemizygous genotypes for Fabry's disease in cultured skin fibroblasts.

Expression of the neutral amino acids transporter ASCT1 in esophageal carcinomas.

Cancers cells utilize more glucose and amino acids than their benign counterparts. Overexpression of the facilitative glucose transporter Glut1 in human cancers was found to correlate with aggressive biologic behavior. The aim of this work was to determine whether the neutral amino acid transporter ASCT1 is expressed in human esophageal carcinomas, and to correlate the findings with Glut1 expression. Sections of formalin-fixed and paraffin-embedded tissue from 42 cases of esophageal carcinomas were entered in the study. Immunohistochemical staining was performed using a rabbit anti-ASCT1 IgG developed in our laboratory, and anti-Glut1 antibody, using standard avidin-streptavidine immunoperoxidase method. Sections of formalin-fixed and paraffin-embedded HepG2 cells were used as positive controls. The percent of ASCT1-positive cells was scored. Statistical analysis was performed using Fisher's exact test. ASCT1 immunoreactivity was cytoplasmic, whereas Glut1 was membranous. Significantly more adenocarcinomas expressed ASCT1 than squamous cell carcinomas (p < 0.0001), whereas Glut1 expression was similar in both tumor types. There was no association between the expression of either transporter and lymph node metastasis. Glut1 was expressed more often in the better differentiated than the poorly differentiated squamous carcinomas (p = 0.003). These results suggest that unlike in squamous cell carcinoma, ASCT1 plays a significant role in the recruitment of amino acids in adenocarcinoma of the esophagus, and suggest that the metabolic needs, and uptake of nutrients, are regulated differently in these two tumor types. Additional studies with larger number of patients are needed to determine the biological significance of ASCT1 expression in esophageal carcinomas.

Accumulation of the insoluble PiZ variant of human alpha 1-antitrypsin within the hepatic endoplasmic reticulum does not elevate the steady-state level of grp78/BiP.

Greater than 85% of the transport-impaired PiZ variant of human alpha 1-antitrypsin is retained within cells and subsequently degraded within a pre-Golgi nonlysosomal compartment that is apparently separate from the endoplasmic reticulum (ER) (Le, A., Graham, K. S., and Sifers, R. N. (1990) J. Biol. Chem. 265, 14001-14007). Despite this phenomenon, human patients and PiZ-bearing transgenic mice exhibit an accumulation of the undegraded protein as insoluble aggregates within distended cisternae of the hepatic ER (Carlson, J. A., Rogers, B. B., Sifers, R. N., Finegold, M. J., Clift, S. M., DeMayo, F. J., Bullock, D. W., and Woo, S. L. C. (1989) J. Clin. Invest. 83, 1183-1190). Immunoprecipitation of the PiZ variant from pulse-radiolabeled hepatocytes from the transgenic animals has demonstrated that a minute quantity of the newly synthesized mutant protein is apparently resistant to degradation and accumulates gradually within the particulate fraction of the cell. Although the steady-state level of the resident ER protein grp78/BiP is elevated in response to the accumulation of malfolded proteins within that subcellular compartment, this phenomenon is not elicited by the accumulation of the insoluble PiZ variant. These results indicate that neither the accumulation of this malfolded protein within the ER nor even the distention of that subcellular compartment is sufficient to cause the up-regulation of grp78/BiP levels. The interpretation of these results with regard to the factors that regulate the levels of grp78/BiP in the ER is discussed.

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.

Genetic control of human alpha-1-antitrypsin.

Alpha-1-antitrypsin (AAT) is the predominant protease inhibitor in human sera. The major physiological role of this inhibitor is to protect elastin fibers in the alveolar structure of the lung from excessive degradation by neutrophil elastase. AAT is synthesized predominantly by hepatocytes, although the AAT gene is expressed to a small degree in the epithelial cells of various tissues. Recent studies have shown that the enhanced liver-specific expression of the AAT gene is controlled by the binding of hepatic nuclear proteins to specific DNA sequences upstream from the structural gene. A variety of mutations within the AAT gene have been identified that result in a partial deficiency or total absence of the inhibitor in sera. Inheritance of a particular combination of these alleles can result in a predisposition towards the development of destructive lung disease. Interestingly, the most common AAT deficiency variant, designated PiZ, causes the mutant protein to accumulate as an insoluble aggregate within the lumen of the hepatic rough endoplasmic reticulum, which is an etiological agent for the development of liver disease. Overall, investigation into the genetic control of AAT has led to an increased understanding of the factors that control hepatic gene expression, as well as mechanisms involved in the pathophysiology of emphysema and liver cirrhosis.

Alpha-1-antitrypsin deficiency: accumulation or degradation of mutant variants within the hepatic endoplasmic reticulum.

Recent molecular and biochemical analyses of several alpha-1-antitrypsin variants suggest that the severe deficiency or complete absence of this protease inhibitor from serum results predominantly from the retention of mutant variants within the hepatic endoplasmic reticulum where they can accumulate or undergo intracellular degradation. Additional studies have demonstrated that the accumulation of the insoluble PiZ variant within this subcellular compartment acts as an etiologic agent for the development of liver disease in transgenic mice.

Molecular biology and genetics of alpha 1-antitrypsin deficiency.

The use of advanced recombinant DNA technology has provided an improved understanding of the human AAT deficiency phenotype by providing the amino acid sequence of several variant proteins and by allowing for the production of various cell and animal models to study the molecular and biochemical components of the retention, degradation, and accumulation of these variants in the hepatic ER. Human AAT deficiency will continue to serve as an excellent model for enhancing our current understanding of mechanisms utilized in regulating protein "traffic" in the ER and in elucidating the pathophysiologic components of AAT-related liver disease.

Disruption of the 290-342 salt bridge is not responsible for the secretory defect of the PiZ alpha 1-antitrypsin variant.

Crystallographic studies have previously suggested that Lys290 forms a salt bridge with Glu342 in the serine protease inhibitor alpha 1-antitrypsin. Disruption of the formation of this structural feature by a Glu to Lys substitution at residue 342 in the PiZ variant has been implicated in causing the defective secretion of this mutant protein from hepatocytes (10-15% of normal). To test the validity of this hypothesis, mutant human alpha 1-antitrypsin cDNA constructs coding for specific amino acid substitutions at residues 290 and 342 were generated and the corresponding mutant proteins were expressed in mouse hepatoma cells. When the potential to form the salt bridge was reestablished by a Lys290 to Glu290 substitution in the PiZ variant, its secretion was increased to only 38% of normal. Furthermore, disruption of this structural feature by a Lys290 to Glu290 substitution in the normal inhibitor failed to reduce the secretion of alpha 1-antitrypsin to the extent observed for the PiZ variant (73% of normal). Finally, substitution of the neutral amino acid Gln at residue 342 only reduced the secretion of alpha 1-antitrypsin to 55% of normal. Of all mutant proteins tested, those bearing Lys at position 342 were secreted at the lowest levels. These findings demonstrate that although disruption of the 290-342 salt bridge does affect the secretion of alpha 1-antitrypsin, it is the substitution of Lys at residue 342 that causes the dramatic secretory defect of the PiZ variant.

Loss of electron-dense lamellar material from Fabry's disease fibroblasts after enzyme replacement.

Cultured fibroblasts from a patient with Fabry's disease were treated with alpha-galactosidase A. The cells internalized the enzyme via a receptor-mediated transport system, resulting in the uptake of enzyme to 50% of the activity of normal cells. Following uptake of the enzyme and incubation for 9 days, a loss of electron-dense lamellar material within membrane-bound residual bodies was detected by electron microscopy. Morphometric analysis of electron micrographs showed that the percentage volume of cytoplasm occupied by electron-dense lamellar material in Fabry's disease fibroblasts decreased to near normal after treatment with enzyme. These results indicate that the ultrastructural abnormalities of Fabry's disease cells can be corrected by enzyme replacement, at least in cultured fibroblasts.

A frameshift mutation results in a truncated alpha 1-antitrypsin that is retained within the rough endoplasmic reticulum.

The major physiological role of the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) is to protect elastic fibers in the lung from excessive hydrolysis by neutrophil elastase. Genetic deficiency of alpha 1-AT predisposes individuals toward the development of emphysema. We have cloned and characterized a mutant alpha 1-AT gene from an individual exhibiting a total absence of immunoreactive alpha 1-AT in serum. Nucleotide sequence analysis of this "null" allele has demonstrated a TC dinucleotide deletion within the codon for Leu318 in exon IV. This frame-shift mutation results in the generation of a premature termination codon at residue 334, which is upstream of the active inhibitory site. To determine the biochemical basis of the null phenotype, the mutant and normal genes were transferred into mouse hepatoma cells for expression analysis. Pulse-chase experiments demonstrated that the mutant gene is expressed into a truncated protein of 45 kDa, which is retained within the rough endoplasmic reticulum. The complete lack of secretion of the truncated protein is consistent with the absence of immunoreactive alpha 1-AT in the patient's serum. In addition, a G to A transition was identified in exon II of the mutant gene, changing the codon for Arg101 to His101. Finally, an A to C transversion was identified in exon V changing the codon for Glu376 to Asp376. Since the latter conservative amino acid substitution has previously been identified in the common PiM2 variant, the frame-shift mutation might have occurred on a PiM2 background chromosome. Using the birthplace of this index case, this mutant alpha 1-AT allele has been designated "nullHong Kong."

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.

Intracellular disposal of incompletely folded human alpha1-antitrypsin involves release from calnexin and post-translational trimming of asparagine-linked oligosaccharides.

Protection of lung elastin fibers from proteolytic destruction is compromised by inefficient secretion of incompletely folded allelic variants of human alpha1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient sedimentation and coimmunoprecipitation techniques were employed to investigate quality control of human alpha1-antitrypsin secretion from stably transfected mouse hepatoma cells. The secretion-incompetent variant null(Hong Kong) (Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335) cannot fold into a functional conformation and was quantitatively associated with the molecular chaperone calnexin following biosynthesis. Assembly with calnexin required cotranslational trimming of glucose from asparagine-linked oligosaccharides. Intracellular disposal of pulse-radiolabeled molecules coincided with their release from calnexin. Released monomers and intracellular disposal were nonexistent in cells chased with cycloheximide, an inhibitor of protein synthesis. Post-translational trimming of asparagine-linked oligosaccharides and intracellular disposal were abrogated by 1-deoxymannojirimycin, an inhibitor of alpha-mannosidase activity, without affecting the monomer population. The data are consistent with a recently proposed quality control model (Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917) in which intracellular disposal requires dissociation from calnexin and post-translational trimming of mannose from asparagine-linked oligosaccharides.

Intracellular association between UDP-glucose:glycoprotein glucosyltransferase and an incompletely folded variant of alpha1-antitrypsin.

Genetic variants of human alpha1-antitrypsin unable to fold into the native structural conformation are poorly secreted from hepatocytes. The molecular chaperone calnexin coimmunoprecipitates with secretion-incompetent variant null(Hong Kong) retained in stably transfected mouse hepatoma cells (Le, A., Steiner, J. L., Ferrell, G. A., Shaker, J. F., and Sifers, R. N. (1994) J. Biol. Chem. 269, 7514-7519). Mobilization of intracellular Ca2+ stores with metabolic poisons diminished interaction with calnexin and coincided with coimmuoprecipitation of a 150-kDa protein (p150). Mobilization of endoplasmic reticulum lumenal Ca2+ with thapsigargin, an inhibitor of the microsomal Ca2+ATPase, gave a similar result. Coimmunoprecipitation of p150 was specifically disrupted in response to incubation of the cell lysate with exogenous CaCl2. Finally, in ECL Western blotting, p150 was recognized by polyclonal antiserum against UDP-glucose:glycoprotein glucosyltransferase that likely functions in glycoprotein folding and quality control (Sousa, M. C., Ferrero-Garcia, M. A., and Parodi, A. J. (1992) Biochemistry 31, 97-105). The data are consistent with a model in which perturbation of endoplasmic reticulum Ca2+ results in a stable physical association between unfolded human alpha1-antitrypsin and UDP-glucose:glycoprotein glucosyltransferase.

Association between calnexin and a secretion-incompetent variant of human alpha 1-antitrypsin.

The naturally occurring nullHong Kong variant of human alpha 1-antitrypsin is truncated at its carboxyl terminus, and is retained and degraded in a pre-Golgi compartment of stably transfected murine hepatoma cells (Le, A., Graham, K. S., and Sifers, R. N. (1990) J. Biol. Chem. 265, 14001-14007). Long-term metabolic radiolabeling with [35S]methionine or [32P]orthophosphate in combination with low stringency immunoprecipitation of the nullHong Kong variant has resulted in the co-precipitation of a radiolabeled 90-kDa protein designated p90. Several criteria, including mobility in SDS-polyacrylamide gel electrophoresis, absence of asparagine-linked oligosaccharides, and immunoreactivity with peptide-specific antiserum, have indicated that co-precipitating p90 is identical to calnexin, a calcium-binding phosphoprotein of the endoplasmic reticulum membrane (Wada, I. W., Rindress, P. H., Ou, W.-J., Doherty, J. J., Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). Finally, results from co-immunoprecipitation analyses and velocity sedimentation experiments have verified that approximately 30% of the retained nullHong Kong variant polypeptides are associated with calnexin in a 1:1 molar ratio and can be dissociated with either deoxycholate or chelation of calcium ions at 37 degrees C. Overall, these findings may extend our current understanding of the molecular pathogenesis of serum alpha 1-antitrypsin deficiency.

Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways.

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded alpha(1)-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.

Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome.

The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human alpha1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded alpha1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded alpha1-antitrypsin for degradation by the proteasome.

Prenatal diagnosis of alpha 1-antitrypsin deficiency by restriction fragment length polymorphisms, and comparison with oligonucleotide probe analysis.

Prenatal diagnosis of sixteen pregnancies at risk for alpha 1-antitrypsin (AAT) deficiency has been achieved by restriction fragment length polymorphism (RFLP) analysis and compared with diagnostic results using hybridisation of M and Z specific oligonucleotides. The results of both tests were in accord for all samples, although under routine laboratory conditions RFLP analysis was more reliable. Because RFLP analysis does not depend on the type of mutation it was possible, in the product of an MZ and SZ mating, to predict an MZ rather than an MS phenotype using the RFLP method. The strong linkage dysequilibrium between an AvaII RFLP and the Z allele increases its diagnostic usefulness. Even so it seems reasonable to use oligonucleotide analysis in families where no siblings are available for comparison. In all other situations RFLP analysis is as accurate and reliable as oligonucleotide analysis and is technically easier, making it the preferred means of diagnosis for informative kindreds.