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Endoplasmin, or GRP94, is an ER-located stress inducible molecular chaperone implicated in the folding and assembly of many proteins. The Arabian one-humped camel lives in an environment of thermal stress, nevertheless is able to encounter the risk of misfolded proteins. Here, the cDNA encoding camel GRP94 was isolated by rapid amplification of cDNA ends. The isolated cDNA contained an open reading frame of 2412 bp encoding a protein of 803 amino acids with predicted molecular mass of 92.5 kDa. Nucleotide and protein BLAST analysis of cGRP94 revealed strong conservation between camel and other domestic mammals. Overexpression of cGRP94 in COS-1 cells revealed multiple isoforms including one N-glycosylated species. Immunofluorescence colocalized cGRP94 with the ER resident protein calnexin. Interestingly, none of the cGRP94 isoforms expressed in CHO cells was N-glycosylated, presumably due to folding determinants that mask the N-glycosylation sites as proposed by in silico modelling. Surprisingly, isoforms of cGRP94 were detected in the culture media of transfected cells indicating that the protein, although an ER resident, also is trafficked and secreted into the exterior milieu. The overall striking structural homologies of GRP94s among mammalian reflect their pivotal role in the ER quality control and protein homeostasis. [ABSTRACT FROM AUTHOR]

Dietary starch is finally converted to glucose for absorption by the small intestine mucosal _-glucosidases (sucrase-isomaltase [SI] and maltase-glucoamylase), and control of this process has health implications. Here, the molecular mechanisms were analyzed associated with starch-triggered maturation and transport of SI. Biosynthetic pulse-chase in Caco-2 cells revealed that the high MW SI species (265 kDa) induced by maltose (an _-amylase starch digestion product) had a higher rate of early trafficking and maturation compared with a glucose-induced SI (245 kDa). The maltose-induced SI was found to have higher affinity to lipid rafts, which are associated with enhanced targeting to the apical membrane and higher activity. Accordingly, in situ maltose-hydrolyzing action was enhanced in the maltose-treated cells. Thus, starch digestion products at the luminal surface of small intestinal enterocytes are sensed and accelerate the intracellular processing of SI to enhance starch digestion capacity in the intestinal lumen.--Chegeni, M., Amiri, M., Nichols, B. L., Naim, H. Y., Hamaker, B. R. Dietary starch breakdown product sensing mobilizes and apically activates _-glucosidases in small intestinal enterocytes. [ABSTRACT FROM AUTHOR]

Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal levels of cellular stress. Since HSPs are key players in stress resistance and protein homeostasis, we aimed to analyze their forms at the cellular and molecular level using camel and human HSPs as models for efficient and moderate thermotolerant mammals, respectively. In this study, we cloned the cDNA encoding two inducible HSP members, HSPA6 and CRYAB from both camel (Camelus dromedarius) and human in a Myc-tagged mammalian expression vector. Expression of these chaperones in COS-1 cells revealed protein bands of approximately 25-kDa for both camel and human CRYAB and 70-kDa for camel HSPA6 and its human homologue. While localization and trafficking of the camel and human HSPs revealed similar cytosolic localization, we could demonstrate altered glycan structure between camel and human HSPA6. Interestingly, the glycoform of camel HSPA6 was rapidly formed and stabilized under normal and stress culture conditions whereas human HSPA6 reacted differently under similar thermal and hypoxic stress conditions. Our data suggest that efficient glycosylation of camel HSPA6 is among the mechanisms that provide camelids with a superior capability for alleviating stressful environmental circumstances. [ABSTRACT FROM AUTHOR]

Background Lactase phlorizin-hydrolase (LPH) is a membrane anchored type I glycoprotein of the intestinal epithelium that is composed of four homologous structural domains. The role of each distinct domain in the intramolecular organization and function of LPH is not completely understood. Methods Here, we analyzed the early events of LPH biosynthesis and trafficking by directed restructuring of the domain compositions. Results Removal of domain I (LPH∆1) results in a malfolded ER-localized protein. By contrast, LPH without domain II (LPH∆2) is normally transported along the secretory pathway, but does not dimerize nor is enzymatically active. Interestingly a polypeptide stretch in domain II between L 735 -R 868 exerts an intriguing role in modulating the trafficking behavior of LPH and its biological function. In fact, association of this stretch with transport-competent LPH chimeras results in their ER-arrest or aberrant trafficking. This stretch harbors a unique N-glycosylation site that is responsible for LPH retention in the ER via association with calnexin and facilitates proper folding of domains I and III before ER exit of LPH. Notably, a similar N-glycosylation site is also found in domain IV with comparable effects on the trafficking of LPH-derived molecules. Conclusions Our study provides novel insights into the intramolecular interactions and the sequence of events involved in the folding, dimerization and transport of LPH. General Significance: Elucidation of the structural-functional relevance of the domains in pro-LPH is crucial in unravelling and understanding the molecular basis of carbohydrate malabsorption disorders that are associated with lactase deficiency or lactase malfunction. [ABSTRACT FROM AUTHOR]