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In JoVE (1)
Other Publications (2)
Articles by Paula E. Magnelli in JoVE
JoVE General
Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases
Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.
Other articles by Paula E. Magnelli on PubMed
A Glucanase-driven Fractionation Allows Redefinition of Schizosaccharomyces Pombe Cell Wall Composition and Structure: Assignment of Diglucan
Purified endoglucanases have been used to determine the composition of Schizosaccharomyces pombe cell wall. This structure has been traditionally studied after isolating its components (mannoproteins, alpha1,3-glucan, beta1,3-glucan, and a branched beta-glucan) with hot alkali. Instead, we sequentially removed the polysaccharides by digesting with endo-beta1,3-glucanase and with a novel endo-alpha1,3-glucanase (mutanase). After this gentle isolation we observed that a branched beta1,3-beta1,6-glucan is much more abundant than previously described. By scaling-up the new protocol we prepared large amounts of the highly branched glucan and determined its structural features. We have named this highly branched beta-glucan diglucan, reflecting its two types of beta linkages. We have also identified an insoluble endoglucanase-resistant type of 1,3-linked glucan present in S. pombe cell walls. We redefined the wall composition of S. pombe vegetative cells by this new method. Finally, to demonstrate its application, we determined the cell wall composition of known mutant strains.
Unique Posttranslational Modifications of Chitin-binding Lectins of Entamoeba Invadens Cyst Walls
Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an approximately 30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans.
