Michael R. Leach

Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non- glycosylated proteins

Calreticulin (CRT) is thought to be a molecular chaperone that interacts with glycoproteins exclusively through a lectin site specific for monoglucosylated oligosaccharides. However, this chaperone function has never been directly demonstrated nor is it clear how lectin–oligosaccharide interactions facilitate glycoprotein folding. Using purified components, we show that CRT suppresses the aggregation not only of a glycoprotein bearing monoglucosylated oligosaccharides but also that of non‐glycosylated proteins. Furthermore, CRT forms stable complexes with unfolded, non‐glycosylated substrates but does not associate with native proteins. ATP and Zn2+ enhance CRTs ability to suppress aggregation of non‐ glycoproteins, whereas engagement of its lectin site with purified oligosaccharide attenuates this function. CRT also confers protection against thermal inactivation and maintains substrates in a folding‐competent state. We conclude that in addition to being a lectin CRT possesses a polypeptide binding capacity capable of discriminating between protein conformational states and that it functions in vitro as a classical molecular chaperone.

The Role of Complex Formation between the Escherichia coli Hydrogenase Accessory Factors HypB and SlyD

The Escherichia coli protein SlyD is a member of the FK-506-binding protein family of peptidylprolyl isomerases. In addition to its peptidylprolyl isomerase domain, SlyD is composed of a molecular chaperone domain and a C-terminal tail rich in potential metal-binding residues. SlyD interacts with the [NiFe]-hydrogenase accessory protein HypB and contributes to nickel insertion during biosynthesis of the hydrogenase metallocenter. This study examines the HypB-SlyD complex and its significance in hydrogenase activation. Protein variants were prepared to delineate the interface between HypB and SlyD. Complex formation requires the HypB linker region located between the high affinity N-terminal Ni(II) site and the GTPase domain of the protein. In the case of SlyD, the deletion of a short loop in the chaperone domain abrogates the interaction with HypB. Mutations in either protein that disrupt complex formation in vitro also result in deficient hydrogenase production in vivo, indicating that the contact between HypB and SlyD is important for hydrogenase maturation. Surprisingly, SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation. Furthermore, a SlyD truncation lacking the C-terminal metal-binding tail still interacts with HypB but is deficient in stimulating metal release and is not functional in vivo. These results suggest that SlyD could activate metal release from HypB during metallation of the [NiFe] hydrogenase.

Lectin-deficient Calnexin Is Capable of Binding Class I Histocompatibility Molecules in Vivo and Preventing Their Degradation

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc1Man9GlcNAc2, calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc1 Man9GlcNAc2 oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean α-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules Kb and Db as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with Kb and Db heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.

Structural and Biological Analysis of the Metal Sites of Escherichia coli Hydrogenase Accessory Protein HypB

The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.

The Peptidyl-Prolyl Isomerase Activity of SlyD Is Not Required for Maturation of Escherichia coli Hydrogenase

Escherichia coli SlyD, which is involved in the biosynthesis of the metal cluster in the [NiFe]-hydrogenase enzymes, exhibits several activities including that of a peptidyl-prolyl isomerase (PPIase). Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo.

Calnexin and Calreticulin, Molecular Chaperones of the Endoplasmic Reticulum

In this chapter we present the evidence that calnexin (CNX) and calreticulin (CRT) function as molecular chaperones to assist in the folding and subunit assembly of the majority of Asn-linked glycoproteins that pass through the endoplasmic reticulum. Mechanistic insights into how this function is accomplished have been provided through diverse approaches which include interfering with the recognition of glycoproteins through CNX/CRT’s lectin site, expression of CNX/CRT and model substrates in heterologous systems, gene disruption, and reconstitution of function with purified components in vitro. Furthermore, the domain organization and locations of functional sites have been revealed through mutagenesis and the recent determination of the structure of the ER luminal domain of CNX and a portion of CRT. The controversial issue of whether CNX/CRT function solely as lectins or also as “classical” chaperones that recognize the unfolded polypeptide portion of glycoproteins is presented and the evidence supporting current models is discussed in detail.

Synthesis of a molecular mimic of the Glc1Man9 oligoside as potential inhibitor of calnexin binding to ΔF508 CFTR protein

Deletion of phenylalanine at position 508 of the CFTR protein is associated with a severe form of cystic fibrosis. Biosynthetic arrest of the misfolded DeltaF508 CFTR protein in the endoplasmic reticulum is due to prolonged interaction with protein chaperones. In order to overcome this retention and thereby restore the delivery of the protein to the plasma membrane, a molecular mimic of the glycoprotein oligoside moiety has been designed and synthesized. Ability of this mimic to inhibit the binding of the natural Glc1Man9GlcNAc oligoside to calnexin has been measured.