Spatial clustering of isozyme-specific residues reveals unlikely determinants of isozyme specificity in fructose-1,6-bisphosphate aldolase

Vertebrate fructose-1,6-bisphosphate aldolase exists as three isozymes (A, B, and C) that demonstrate kinetic properties that are consistent with their physiological role and tissue-specific expression. The isozymes demonstrate specific substrate cleavage efficiencies along with differences in the ability to interact with other proteins; however, it is unknown how these differences are conferred. An alignment of 21 known vertebrate aldolase sequences was used to identify all of the amino acids that are specific to each isozyme, or isozyme-specific residues (ISRs). The location of ISRs on the tertiary and quaternary structures of aldolase reveals that ISRs are found largely on the surface (24 out of 27) and are all outside of hydrogen bonding distance to any active site residue. Moreover, ISRs cluster into two patches on the surface of aldolase with one of these patches, the terminal surface patch, overlapping with the actin-binding site of aldolase A and overlapping an area of higher than average temperature factors derived from the x-ray crystal structures of the isozymes. The other patch, the distal surface patch, comprises an area with a different electrostatic surface potential when comparing isozymes. Despite their location distal to the active site, swapping ISRs between aldolase A and B by multiple site mutagenesis on recombinant expression plasmids is sufficient to convert the kinetic properties of aldolase A to those of aldolase B. This implies that ISRs influence catalysis via changes that alter the structure of the active site from a distance or via changes that alter the interaction of the mobile C-terminal portion with the active site. The methods used in the identification and analysis of ISRs discussed here can be applied to other protein families to reveal functionally relevant residue clusters not accessible by conventional primary sequence alignment methods.     

Metabolic Compartmentation in Living Cells: Structural Association of Aldolase

The glycolytic enzyme aldolase is concentrated in a domain around stress fibers in living Swiss 3T3 cells, but the mechanism by which aldolase is localized has not been revealed. We have recently identified a molecular binding site for F-actin on aldolase, and we hypothesized that this specific binding interaction, rather than a nonspecific mechanism, is responsible for localizing aldolasein vivo.In this report, we have used fluorescent analog cytochemistry of a site-directed mutant of aldolase to demonstrate that actin-binding activity localizes this molecule along stress fibers in quiescent cells and behind active ruffles in the leading edge of motile cells. The specific cytoskeletal association of aldolase could play a structural role in cytoplasm, and it may contribute to metabolic regulation, metabolic compartmentation, and/or cell motility. Functional duality may be a widespread feature among cytosolic enzymes.     

Technical comment on Boersma et al. (2016) Temperature driven changes in the diet preference of omnivorous copepods: no more meat when it's hot? Ecology Letters, 19, 45–53

A recent study concluded that omnivorous plankton will shift from predatory to herbivorous feeding with climate warming, as consumers require increased carbon:phosphorous in their food. Although this is an appealing hypothesis, we suggest the conclusion is unfounded, based on the data presented, which seem in places questionable and poorly interpreted.     

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N - acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.      

Topological studies on the enzymes catalyzing the biosynthesis of Glc-P- dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers

In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2- P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face of the ER and diffuse transversely to the lumenal leaflet where the synthesis of the lipid-bound precursor oligosaccharide is completed. To establish the topological sites of Glc-P-Dol synthesis and the lipid-mediated glucosyltransfer reactions involved in Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P- Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined in sealed microsomal vesicles. Since ER vesicles from brain do not contain glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the lumenally oriented, processing glucosidase I/II activities was used to assess the intactness of the vesicle preparations. Comparative enzymatic studies with sealed ER vesicles from brain and kidney, a tissue that contains Glc 6-P phosphatase, demonstrate the reliability of using the processing glucosidase activities as latency markers for topological studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc 6-P phosphatase. The results obtained from the trypsin-sensitivity assays with sealed microsomal vesicles from brain are consistent with a topological model in which Glc-P-Dol is synthesized on the cytoplasmic face of the ER, and subsequently utilized by the three Glc-P-Dol-mediated GlcTases after "flip-flopping" to the lumenal monolayer.      

A feature selection approach for identification of signature genes from SAGE data

Background  

One goal of gene expression profiling is to identify signature genes that robustly distinguish different types or grades of tumors. Several tumor classifiers based on expression profiling have been proposed using microarray technique. Due to important differences in the probabilistic models of microarray and SAGE technologies, it is important to develop suitable techniques to select specific genes from SAGE measurements.