Influence of Trp flipping on carbohydrate binding in lectins. An example on Aleuria aurantia lectin AAL.

Protein-carbohydrate interactions are very often mediated by the stacking CH-π interactions involving the side chains of aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) or phenylalanine (Phe). Especially suitable for stacking is the Trp residue. Analysis of the PDB database shows Trp stacking for 265 carbohydrate or carbohydrate like ligands in 5 208 Trp containing motives. An appropriate model system to study such an interaction is the AAL lectin family where the stacking interactions play a crucial role and are thought to be a driving force for carbohydrate binding. In this study we present data showing a novel finding in the stacking interaction of the AAL Trp side chain with the carbohydrate. High resolution X-ray structure of the AAL lectin from Aleuria aurantia with α-methyl-l-fucoside ligand shows two possible Trp side chain conformations with the same occupation in electron density. The in silico data shows that the conformation of the Trp side chain does not influence the interaction energy despite the fact that each conformation creates interactions with different carbohydrate CH groups. Moreover, the PDB data search shows that the conformations are almost equally distributed across all Trp-carbohydrate complexes, which would suggest no substantial preference for one conformation over another.

Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma.

The Aleuria aurantia lectin (AAL) derived from orange peel fungus contains five fucose-binding sites that recognizes fucose bound in α-1,2, α-1,3, α-1,4, and α-1,6 linkages to N-acetylglucosamine and galactose. Recently, we have created several recombinant AAL (rAAL) proteins that had altered binding affinity to fucose linkages. In this report, we further characterize the binding specificity of one of the mutated lectins, N224Q lectin. This lectin was characterized by lectin Western blotting, surface plasmon resonance, and glycan microarray and shown to have increased binding to fucosylated glycan. Subsequently, we used this lectin to identify secreted fucosylated glycoproteins from a fetal hepatic cell line. Proteomic analysis revealed several glycoproteins secreted by the fetal cell line that were bound by N224Q lectin. These findings were confirmed by subsequent proteomic analysis of human serum from control patients or patients with hepatocellular carcinoma. These represent candidate oncofetal markers for liver cancer.

Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues.

A new matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in tissues is described. Application of an endoglycosidase, peptide N-glycosidase F (PNGaseF), directly on tissues followed by incubation releases N-linked glycan species amenable to detection by MALDI-IMS. The method has been designed to simultaneously profile the multiple glycan species released from intracellular organelle and cell surface glycoproteins, while maintaining histopathology compatible preparation workflows. A recombinant PNGaseF enzyme was sprayed uniformly across mouse brain tissue slides, incubated for 2 h, then sprayed with 2,5-dihydroxybenzoic acid matrix for MALDI-IMS analysis. Using this basic approach, global snapshots of major cellular N-linked glycoforms were detected, including their tissue localization and distribution, structure, and relative abundance. Off-tissue extraction and modification of glycans from similarly processed tissues and further mass spectrometry or HPLC analysis was done to assign structural designations. MALDI-IMS has primarily been utilized to spatially profile proteins, lipids, drug, and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis. The translatable MALDI-IMS glycan profiling workflow described herein can readily be applied to any tissue type of interest. From a clinical diagnostics perspective, the ability to differentially profile N-glycans and correlate their molecular expression to histopathological changes can offer new approaches to identifying novel disease related targets for biomarker and therapeutic applications.

Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans.

Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and solution based assays. AAL is a mushroom derived lectin that contains five fucose binding sites that preferentially bind fucose linked (α-1,3, α-1,2, α-,4, and α-1,6) to N-acetyllactosamine related structures. Recently, several reports by us and by others have indicated that specific fucose linkages found on certain serum biomarker glycoprotein's are more associated with disease than others. Taking a site-directed mutagenesis approach, we have created a set of recombinant AAL proteins that display altered binding affinities to different analytes containing various fucose linkages.

GP73, a resident Golgi glycoprotein, is a novel serum marker for hepatocellular carcinoma.

BACKGROUND/AIMS: Golgi protein-73 (GP73) is up-regulated in hepatocellular carcinoma (HCC). The aims of this study were to determine if GP73 is detected in the serum, and to establish the sensitivity and specificity of serum GP73 for diagnosing HCC. METHODS: Serum GP73 was detected by immunoblots and quantified by densitometric analysis. RESULTS: A total of 352 patients were studied. Serum GP73 levels were significantly higher in patients with HCC compared to those with cirrhosis (P < 0.001). GP73 had a sensitivity of 69% and a specificity of 75% at the optimal cutoff point of 10 relative units, with an area under the receiver operating curve of 0.79 vs. 0.61 for AFP (P = 0.001). GP73 levels had significantly higher sensitivity (62%) than AFP (25%) for diagnosing early HCC (P < 0.0001). Moreover, GP73 levels were elevated in the serum of 57% (32/56) of individuals with HCC who had serum AFP levels less than 20ng/ml. CONCLUSIONS: Higher levels of GP73 can be found in the serum of patients with HCC than of those without. GP73 was better than AFP for the diagnosis of early HCC. Further validation studies are needed to confirm the role of GP73 in the early detection of HCC.

Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans.

Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core alpha-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.

Comparative proteomic analysis of de-N-glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status.

Analysis of the polypeptide profile in tissues, cells, and sera by high-resolution two-dimensional (2-D) gel electrophoresis offers promise in the identification of biomarkers that correlate with disease. However, sera contain many polypeptides bearing N-linked glycosylation that can complicate interpretation. Therefore, we tested the possibility that de-N-glycosylation of the polypeptides present in human serum would result in a simplification of serum proteome profiles. Briefly, polypeptides present in human serum were left untreated or subjected to de-N-glycosylation by incubation with PNGase F and resolved by high-resolution 2-D gel electrophoresis. De-N-glycosylation reduced the number of glycoform variants, enhanced the resolution of many polypeptides and allowed other polypeptides to become visible. As an initial test of concept, clinically relevant serum samples from individuals with or without diagnosis of hepatocellular carcinoma were compared. Several polypeptides, apparent only after de-N-glycosylation, were shown to correlate with disease. Although the results are preliminary and the identities of all the putative biomarkers not yet known, the data suggest that de-N-glycosylation offers a method to enhance the resolution of serum polypeptide profiles and has value in comparative proteomic studies.

Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2alpha kinase PERK.

The hepatitis C virus envelope protein, E2, is an endoplasmic reticulum (ER)-bound protein that contains a region of sequence homology with the double-stranded RNA-activated protein kinase PKR and its substrate, the eukaryotic translation initiation factor 2 (eIF2). We previously reported that E2 modulates global translation through inhibition of the interferon-induced antiviral protein PKR through its PKR-eIF2alpha phosphorylation site homology domain (PePHD). Here we show that the PKR-like ER-resident kinase (PERK) binds to and is also inhibited by E2. At low expression levels, E2 induced ER stress, but at high expression levels, and in vitro, E2 inhibited PERK kinase activity. Mammalian cells that stably express E2 were refractory to the translation-inhibitory effects of ER stress inducers, and E2 relieved general translation inhibition induced by PERK. The PePHD of E2 was required for the rescue of translation that was inhibited by activated PERK, similar to our previous findings with PKR. Here we report the inhibition of a second eIF2alpha kinase by E2, and these results are consistent with a pseudosubstrate mechanism of inhibition of eIF2alpha kinases. These findings may also explain how the virus promotes persistent infection by overcoming the cellular ER stress response.

Replication of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces endoplasmic reticulum stress-mediated apoptosis of MDBK cells.

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca(2+) homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the Flaviviridae family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2 alpha, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.

P52rIPK regulates the molecular cochaperone P58IPK to mediate control of the RNA-dependent protein kinase in response to cytoplasmic stress.

The 52 kDa protein referred to as P52(rIPK) was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52(rIPK) and P58(IPK) each possess structural domains implicated in stress signaling, including the charged domain of P52(rIPK) and the tetratricopeptide repeat (TPR) and DnaJ domains of P58(IPK). The P52(rIPK) charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52(rIPK) function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58(IPK). This domain bound specifically to P58(IPK) TPR domain 7, the domain adjacent to the TPR motif required for P58(IPK) interaction with PKR, thus providing a mechanism for P52(rIPK) inhibition of P58(IPK) function. Both the charged domain of P52(rIPK) and the TPR 7 domain of P58(IPK) were required for P52(rIPK) to mediate downstream control of PKR activity, eIF2alpha phosphorylation, and cell growth. Furthermore, we found that P52(rIPK) and P58(IPK) formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52(rIPK) charged domain functions as a TPR-specific signaling motif to directly regulate P58(IPK) within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.