N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.     

Use of a structured kinetic model of antibody synthesis and secretion for optimization of antibody production systems: II. Transient analysis

The dynamic behavior of the monoclonal antibody (MAb) secretory pathway is studied by transient simulations using our previously developed structured kinetic model for antibody synthesis and secretion by hybridoma cells. The response of the secretory pathway to blocks in specific pathway steps and step changes in characteristic pathway parameters is presented in order to gain a better understanding of pathway dynamics and identify possible ratelimiting steps in the pathway. Model simulations suggest that the step of antibody assembly in the endoplasmic reticulum (ER) is a very good candidate for a rate-limiting step in the antibody secretory pathway in fast-growing hybridoma cells, whereas translation of the heavy and light chains is most likely rate-limiting in slowly growing or stationary phase cells. Transient simulation results are compared with experimentally observed transient changes in specific antibody secretion rates and used to suggest strategies for optimizing antibody secretion in large-scale production systems.     

Control of glutathione and phytochelatin synthesis under cadmium stress. Pathway modeling for plants

Glutathione (GSH) plays several roles in cell metabolism such as redox state regulation, oxidative stress control, and protection against xenobiotics and heavy metals. GSH is synthesized in two steps catalysed by gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase. gamma-ECS is feedback inhibited by GSH, which has led to the proposal that this enzyme acts as the rate-limiting step in the pathway. Thus far, the study of GSH metabolism has been confined to GSH synthesis (GSH supply), without considering the GSH-consuming enzymes (GSH demand). Several works have shown that the demand block of enzymes may have a significant control on a pathway; therefore, we hypothesize that GSH-consuming enzymes may exert some control on GSH synthesis. A kinetic model of GSH and phytochelatin synthesis in plants was constructed using the software GEPASI and the kinetic data available in the literature. The main conclusions drawn by the model concerning metabolic control analysis are (1) gamma-ECS is indeed a rate-limiting step in GSH synthesis, but only if GSH-consuming enzymes are not taken into account. (2) At low demand, GSH-consuming enzymes exert significant flux-control on GSH synthesis whereas at high demand, supply and demand blocks share the control of flux. (3) In unstressed conditions, flux to GSH is controlled mainly by demand, so that gamma-ECS determines the degree of homeostasis of the GSH concentration. Under cadmium exposure, the GSH demand increases and flux-control is re-distributed almost equally between the supply and demand blocks. (4) To enhance phytochelatins synthesis without depleting the GSH pool, at least two enzymes (gamma-ECS and PCS) should be increased and/or, alternatively, a branching flux (GSH-S-transferases) could be partially diminished.     

Potential folding-function interrelationship in proteins

The possibility is addressed that protein folding and function may be related via regions that are critical for both folding and function. This approach is based on the building blocks folding model that describes protein folding as binding events of conformationally fluctuating building blocks. Within these, we identify building block fragments that are critical for achieving the native fold. A library of such critical building blocks (CBBs) is constructed. Then, it is asked whether the functionally important residues fall in these CBB fragments. We find that for over two-thirds of the proteins in our library with available functional information, the catalytic or binding site residues lie within the CBB regions. From the evolutionary standpoint, a folding-function relationship is advantageous, since the need to guard against mutations is limited to one region. Furthermore, conformationally similar CBBs are found in globally unrelated proteins with different functions. Hence, substituting CBBs may lead to designed proteins with altered functions. We further find that the CBBs in our library are conformationally unstable.     

Protein folding: binding of conformationally fluctuating building blocks via population selection.

Here we review different aspects of the protein folding literature. We present a broad range of observations, showing them to be consistent with a general hierarchical protein folding model. In such a model, local relatively stable, conformationally fluctuating building blocks bind through population selection, to yield the native state. The model includes several components: (1) the fluctuating building blocks that constitute local minima along the polypeptide chain, which even if unstable still possess higher population times than all alternate conformations; (2) the landscape around the bottom of the funnels; (3) the consideration that protein folding involves intramolecular recognition; (4) similar landscapes are observed for folding and for binding, and that (5) the landscape is dynamic, changing with the conditions. The model considers protein folding to be guided by native interactions. The reviewed literature includes the effects of changing the conditions, intermediates and kinetic traps, mutations, similar topologies, fragment complementation experiments, fragments and pathways, focusing on one specific well-studied example, that of the dihydrofolate reductase, chaperones, and chaperonines, in vivo vs. in vitro folding, still using the dihydrofolate example, amyloid formation, and molecular "disorder". These are consistent with the view that binding and folding are similar events, with the differences stemming from different stabilities and hence population times.     

Production of secretory and extracellular N-linked glycoproteins in Escherichia coli

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.     

Pivotal role of calnexin and mannose trimming in regulating the endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain

We have established a mammalian semipermeabilized cell system that faithfully reconstitutes the proteasome-mediated degradation of major histocompatibility complex Class I heavy chain. We show that degradation required unfolding of the protein and was cytosol- and ATP-dependent and that dislocation and degradation required proteasome activity. When the interaction of heavy chain with calnexin was prevented, the rate of degradation was accelerated, suggesting that an interaction with calnexin stabilized heavy chain. Stabilization of heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure degradation of heavy chain. When degradation or mannose trimming was inhibited, heavy chain formed a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway.     

Signal sequences initiate the pathway of maturation in the endoplasmic reticulum lumen

An interaction between an N-terminal signal sequence and the translocon leads to the initiation of protein translocation into the endoplasmic reticulum lumen. Subsequently, folding and modification of the substrate rapidly ensue. The close temporal coordination of these processes suggests that they may be structurally and functionally coordinated as well. Here we show that information encoded in the hydrophobic domain of a signal sequence influences the timing and efficiency of at least two steps in maturation, namely N-linked glycosylation and signal sequence cleavage. We demonstrate that these consequences correlate with and likely stem from the nature of the initial association made between the signal sequence and the translocon during the initiation of translocation. We propose a model by which these maturational events are controlled by the signal sequence-translocon interaction. Our work demonstrates that the pathway taken by a nascent chain through post-translational maturation depends on information encoded in its signal sequence.     

Biogenesis of CFTR and other Polytopic Membrane Proteins: New Rolesfor the Ribosome-Translocon Complex

Polytopic protein biogenesis represents a critical, yet poorly understood area of modern biology with important implications for human disease. Inherited mutations in a growing array of membrane proteins frequently lead to improper folding and/or trafficking. The cystic fibrosis transmembrane conductance regulator (CFTR) is a primary example in which point mutations disrupt CFTR folding and lead to rapid degradation in the endoplasmic reticulum (ER). It has been difficult, however, to discern the mechanistic principles of such disorders, in part, because membrane protein folding takes place coincident with translation and within a highly specialized environment formed by the ribosome, Sec61 translocon, and the ER membrane. This ribosome-translocon complex (RTC) coordinates the synthesis, folding, orientation and integration of transmembrane segments across and into the ER membrane. At the same time, RTC function is controlled by specific sequence determinants within the nascent polypeptide. Recent studies of CFTR and other native membrane proteins have begun to define novel variations in translocation pathways and to elucidate the specific steps that establish complex topology. This article will attempt to reconcile advances in our understanding of protein biogenesis with emerging models of RTC function. In particular, it will emphasize how information within the nascent polypeptide is interpreted by and in turn controls RTC dynamics to generate the broad structural and functional diversity observed for naturally occurring membrane proteins.Abbreviations: AQP, aquaporin; CFTR, cystic fibrosis transmembrane conductance regulator; ECL, extracellular loop; EM, electron microscopy; ER, endoplasmic reticulum; ICL, intracellular loop; PTC, peptidyltransferase center; RNC, ribosome-nascent chain; RTC, ribosome-translocon complex; SRP, signal recognition particle; SR, SRP receptor; TM, transmembrane (segment); TMD, transmembrane domain. ABC, ATP binding cassette; BiP, heavy chain binding protein; FRET, Förster resonance energy transfer; NBD, nucleotide binding domain; SPC, signal peptidase complex; TrAF, translocation-associated factors; TRAM, translocating chain-associated membrane protein; TRAP, translocon-associated protein.     

The effect of glycosylation on the folding kinetics of erythropoietin

Glycosylation is a common posttranslational modification that generally increases protein solubility and thermodynamic stability. Less is known about how this modification influences protein folding, particularly folding processes involving intermediate species. In the present report, folding comparisons of a nonglycosylated erythropoietin (EPO) mutant are made with the fully glycosylated EPO, which was recently shown to fold by a three-state on-pathway mechanism. The absence of glycosylation did not alter the folding mechanism of EPO but did greatly decrease the stability of the intermediate species, change the rate-limiting step of the folding reaction, and accelerate the folding kinetics to both the intermediate state and the native state. Surprisingly, glycosylation stabilized the intermediate species to a greater extent than it increased the EPO equilibrium stability. These results suggest that glycosylation impedes the latter EPO folding steps rather than accelerating them by biasing particular folding pathways, as previously proposed for folding reactions initiated from unfolded ensembles with minimal residual structure. Due to the specific biological processes modulated by EPO glycosylation, however, there may be little evolutionary pressure to fold on a faster, more direct pathway at the expense of biological function, particularly given the protective role glycosylation has at preventing EPO aggregation. Lastly, evidence that is consistent with glycosylation destabilizing the unfolded state to some degree and contributing to the greater equilibrium stability of the glycosylated EPO is presented.     

Identification of specific glycoforms of major histocompatibility complex class I heavy chains suggests that class I peptide loading is an adaptation of the quality control pathway involving calreticulin and ERp57

Glycosylation analysis was used to probe the sequence of events accompanying the binding of antigenic peptides to the major histocompatibility complex class I heavy chains. Free heavy chains were isolated from the beta(2)-microglobulin-negative cell line Daudi and from the B-lymphoblastoid cell line Raji. Heavy chains were also isolated from Raji cells in multimolecular complexes (peptide loading complexes) containing the transporter associated with antigen processing, tapasin and ERp57 with and without the lectin-like folding chaperone, calreticulin. Calreticulin is a soluble protein that recognizes primarily the terminal glucose of Glc(1)Man(7-9)GlcNAc(2) glycans. This paper shows that monoglucosylated glycoforms of heavy chain, which exist transiently in the endoplasmic reticulum in the initial stages of the glycosylation processing pathway, are present in the peptide loading complex. The data are consistent with a model in which the release of peptide-loaded major histocompatibility complex class I molecules from calreticulin, induced by deglucosylation of the heavy chain N-linked glycan, signals the dissociation of the complex. This is consistent with the hypothesis that the class I loading process is an adaptation of the quality control mechanism involving calreticulin and ERp57.     

Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.     

Biogenesis of respiratory cytochromes in bacteria.

Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible.     

An endoplasmic reticulum storage disease causing congenital goiter with hypothyroidism

In humans, deficient thyroglobulin (Tg, the thyroid prohormone) is an important cause of congenital hypothyroid goiter; further, homozygous mice expressing two cog/cog alleles (linked to the Tg locus) exhibit the same phenotype. Tg mutations might affect multiple different steps in thyroid hormone synthesis; however, the microscopic and biochemical phenotype tends to involve enlargement of the thyroid ER and accumulation of protein bands of M(r) < 100. To explore further the cell biology of this autosomal recessive illness, we have examined the folding and intracellular transport of newly synthesized Tg in cog/cog thyroid tissue. We find that mutant mice synthesize a full-length Tg, which appears to undergo normal N-linked glycosylation and glucose trimming. Nevertheless, in the mutant, Tg is deficient in the folding that leads to homodimerization, and there is a deficiency in the quantity of intracellular Tg transported to the distal portion of the secretory pathway. Indeed, we find that the underlying disorder in cog/cog mice is a thyroid ER storage disease, in which a temperature- sensitive Tg folding defect, in conjunction with normal ER quality control mechanisms, leads to defective Tg export. In relation to quality control, we find that the physiological response in this illness includes the specific induction of five molecular chaperones in the thyroid ER. Based on the pattern of chaperone binding, different potential roles for individual chaperones are suggested in glycoprotein folding, retention, and degradation in this ER storage disease.     

Binding and folding: in search of intramolecular chaperone-like building block fragments

We propose an intramolecular chaperone which catalyzes folding and neither dissociates nor is cleaved. This uncleaved foldase is an intramolecular chain-linked chaperone, which constitutes a critical building block of the structure. Macroscopically, all molecular chaperones facilitate folding reactions and manifest similar energy landscapes. However, microscopically they differ. While intermolecular chaperones catalyze folding by unfolding misfolded conformations or prevent misfolding, the chain-linked cleaved (proregion) and uncleaved intramolecular chaperone-like building blocks suggested here, catalyze folding by binding to, stabilizing and increasing the populations of native conformations of adjacent building block fragments. In both, the more stable the intramolecular chaperone fragment region, the faster is the folding rate. Hence, mechanistically, intramolecular chaperones and chaperone-like segments are similar. Both play a dual role, in folding and in protein function. However, while the functional role of the proregions is inhibitory, necessitating their cleavage, the function of the uncleaved intramolecular chaperone-like building blocks does not require their subsequent removal. On the contrary, it requires that they remain in the structure. This may lead to the difference in the type of control they are under: proteins folding with the assistance of the proregion have been shown to be under kinetic control. It has been suggested that kinetically controlled folding reactions, with the proregion catalyst removed, lend longevity under harsh conditions. On the other hand, proteins with uncleaved intramolecular chaperone-like building blocks, with their 'foldases' still attached, are largely under thermodynamic control, consistent with the control observed in most protein folding reactions. We propose that an uncleaved intramolecular chaperone-like fragment occurs frequently in proteins. We further propose that such proteins would be prone to changing conditions and in particular, to mutations in this critical building block region. We describe the features qualifying it for its proposed chaperone-like role, compare it with inter- and intramolecular chaperones and review current literature in this light. We further propose a mechanism showing how it lowers the barrier heights, leading to faster folding reaction rates. Since these fragments constitute an intergal part of the protein structure, we call these critical building blocks intramolecular, chaperone-like fragments, to clarify, distinguish and adhere to the definition of the transiently associating chaperones. The new mechanism presented here differs from the concept of 'folding nuclei'. While the concept of folding nuclei focuses on a non-sequential distribution of the folding information along the entire protein chain, the chaperone-like building block fragments proposition focuses on a segmental distribution of the folding information. This segmental distribution controls the distributions of the populations throughout the hierarchical folding processes.     

Protein-specific features of the general secretion pathway in yeast: the secretion of acid phosphatase

The major phosphate-repressible acid phosphatase (APase) of Saccharomyces cerevisiae, a cell wall glycoprotein, has been extensively used as a reporter protein to analyse successive steps in the yeast secretory pathway. In contrast to other yeast secretory proteins, APase can still be translocated into the endoplasmic reticulum (ER) even when it is made without its signal peptide. This property illustrates the permissiveness of targeting to the ER in yeast. Studies on APase-containing hybrid proteins have provided some of the evidence that specific soluble factors must interact with secretory proteins prior to their translocation across the ER membrane. A systematic analysis of mutations affecting the sequence of the APase signal peptide cleavage site demonstrated that cleavage occurs only when the last amino acid of the signal sequence is small and neutral. This was one of the first studies to verify the requirements for signal peptidase cleavage that had previously only been predicted from statistical analysis. Studies performed either with inhibitors of glycosylation or with mutant APases demonstrated the critical role of core glycosylation for APase folding, which is essential for efficient transport beyond the ER. Following the fate of particular modified APases along the secretory pathway provided insights into some general properties of the secretory apparatus and illustrated the specific requirements for a given protein during its intracellular traffic.     

Protein folding and protein refolding.

The functional three-dimensional structure of proteins is determined solely by their amino acid sequences. Protein folding occurs spontaneously beginning with the formation of local secondary structure concomitant with a compaction of the molecule. Secondary structure elements subsequently interact to form subdomains and domains stabilized by tertiary interactions. Disulfide bond formation, and cis-trans isomerization of X-Pro peptide bonds, as the rate-limiting folding reactions, are enzymatically catalyzed during protein folding in the cell. Although folding of domains is fast enough to occur cotranslationally in vivo, such vectorial folding on the ribosome is not essential for attainment of the native structure of a protein. Slow steps on the pathway to the functional protein structure are docking reactions of domains, association of subunits, or reshuffling reactions at the oligomer level. Aggregation as a competing side reaction is prevented, and the kinetic partition between competing polypeptide folding and translocation reactions is regulated by chaperone proteins binding to incompletely folded polypeptides.