Chapter one - Analysis of Serpin Secretion, Misfolding, and Surveillance in the Endoplasmic Reticulum
Introduction
The conformational maturation and secretion of an active serpin involves a multistep process. Initially, cellular machinery orchestrate a series of requisite events that eventually direct the cotranslational insertion of the nascent polypeptide across the membrane of the rough endoplasmic reticulum (ER) and into its lumen (Cabral et al., 2001). Transient physical interactions with a group of molecular chaperones promote correct protein folding by diminishing the frequency of unproductive folding events that can lead to inappropriate aggregation (Choudhury et al., 1977, Fewell et al., 2001).
Efficient conformational maturation functions as a quality control standard, permitting the progression of active proteins beyond the ER (Wu et al., 2003). Noncompliance results in the ER retention of misfolded polypeptides and unassembled protein subunits, both of which exhibit prolonged physical interaction with molecular chaperones and are eventually targeted for intracellular degradation (Liu et al., 1997). In recent years, a picture has emerged in which the addition of asparagine-linked oligosaccharides, and their covalent modification, functions to assist the productive folding of newly synthesized serpins by facilitating their physical interactions with a small family of lectins that contribute to the glycoprotein folding machinery (Cabral et al., 2001, Ellgaard et al., 1999).
Importantly, additional carbohydrate modifications are responsible for generating a tag that selectively targets the nonnative proteins for intracellular degradation (Cabral et al., 2001, Molinari, 2007). A model has been proposed in which the timing of the latter glycan modification, relative to the duration of nonnative glycoprotein structure, functions as an underlying stochastic mechanism by which serpins that exhibit permanent structural defects are selectively targeted for intracellular disposal (Wu et al., 2003). Later stages of the disposal process include retrotranslocation across the ER membrane and into the cytoplasm, polyubiquitination, and elimination by 26S proteasomes (Bonifacino and Weissman, 1998).
Almost all naturally occurring secretion-incompetent serpin variants misfold following biosynthesis (Sifers et al., 1992), and therefore fail to pass the quality control checkpoint. The serpins have provided a model system to elucidate how glycoprotein processing contributes to these surveillance mechanisms. The following information describes the experimental strategies and methodologies designed to elucidate the involvement of asparagine-linked oligosaccharide processing in regulating the fate of a newly synthesized serpin.
Section snippets
Transfection of mammalian cell lines
Because the biosynthesis of serpins is naturally expressed in only a few cell types, it will be necessary to transfect the serpin cDNA into a selected cultured mammalian cell line to generate a system for studying the protein's fate following biosynthesis. The wild-type cDNA can be utilized in the expression studies, or one that has undergone site-directed mutagenesis either to mimic a naturally occurring variant or to test a specific structural hypothesis. The cDNA is subcloned into any number
Concluding Remarks
Very little is known, or even appreciated, as to how biological systems that function exclusively at the level of encoded proteins can contribute to, or modify, gene expression. Conceivably, human biological variation at this level can influence the phenotypic expression of an individual's genotype (Cabral et al., 2002). The devised methodologies outlined above describe various experimental strategies that have successfully elucidated the roles played by asparagine-linked oligosaccharide
Acknowledgments
We acknowledge funding (to R. N. S.) by multiple grants from the National Institutes of Health (NIHLB and NIDDK), American Lung Association, American Heart Association, Alpha1-Foundation, and the Moran Foundation. S. P. was funded by a postdoctoral fellowship from the Alpha1-Foundation and a grant from the Baylor College of Medicine Digestive Disease Center (DK56338). M. J. I. is the recipient of a training grant from the Huffington Center on Aging, at Baylor College of Medicine.
References (38)
- et al.
Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways
J. Biol. Chem.
(2000) - et al.
Dissecting glycoprotein quality control in the secretory pathway
Trends Biochem. Sci.
(2001) - et al.
ER quality control: Towards an understanding at the molecular level
Curr. Opin. Cell Biol.
(2001) - et al.
Accumulation of the insoluble PI Z variant of human α1-antitrypsin within the hepatic endoplasmic reticulum does not elevate the steady-state level of grp78/BiP
J. Biol. Chem.
(1990) - et al.
Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum
Cell
(1995) - et al.
Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I
J. Biol. Chem.
(2003) - et al.
Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases
Biochem. Biophys. Res. Commun.
(2007) - et al.
Intracellular degradation of the transport-impaired human PI Z α1-antitrypsin variant. Biochemical mapping of the degradative event among compartments of the secretory pathway
J. Biol. Chem.
(1990) - et al.
Soluble aggregates of the human PI Z α1-antitrypsin variant are degraded within the endoplasmic reticulum by a mechanism sensitive to inhibitors of protein synthesis
J. Biol. Chem.
(1992) - et al.
Association between calnexin and a secretion-incompetent variant of human α1-antitrypsin
J. Biol. Chem.
(1994)
Intracellular disposal of incompletely folded human α1-antitrypsin involves release from calnexin and post-translational trimming of asparagine-linked oligosaccharides
J. Biol. Chem.
Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome
J. Biol. Chem.
Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator
J. Biol. Chem.
Alpha1-antitrypsin as a model for glycan function in the endoplasmic reticulum
Methods
Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis
J. Biol. Chem.
Ubiquitin and the control of protein fate in the secretory and endocytic pathways
Annu. Rev. Cell Dev. Biol.
Organizational diversity among distinct glycoprotein ER-associated degradation programs
Mol. Biol. Cell
Intracellular association between udp-glucose:glycoprotein glucosyltransferase and an incompletely folded variant of α1-antitrypsin
J. Biol. Chem.
Quality control of protein folding: Participation in human disease
News Physiol. Sci.
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