Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase

Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase.     

What company characteristics are associated with the adoption of social media?

ABSTRACT

In just one decade, social media have moved from being marginal phenomena to having three billion active monthly users globally in 2017. We use a large representative sample to study which company characteristics are associated with the early adoption of social media. Our findings suggest that digital orientation (outside social media), innovativeness, external collaboration in marketing and sales, and orientation towards consumer markets all increase a company’s probability of adopting social media. In addition, larger and younger companies are more likely to be early movers. Overall, business use of social media has only reached its early maturity; thus, it still has plenty of room to grow.     

Activation and oligomerization of aspartylglucosaminidase

Secretory, membrane, and lysosomal proteins undergo covalent modifications and acquire their secondary and tertiary structure in the lumen of the endoplasmic reticulum (ER). In order to pass the ER quality control system and become transported to their final destinations, many of them are also assembled into oligomers. We have recently determined the three-dimensional structure of lysosomal aspartylglucosaminidase (AGA), which belongs to a newly discovered family of homologous amidohydrolases, the N-terminal nucleophile hydrolases. Members of this protein family are activated from an inactive precursor molecule by an autocatalytic proteolytic processing event whose exact mechanism has not been thoroughly determined. Here we have characterized in more detail the initial events in the ER required for the formation of active AGA enzyme using transient expression of polypeptides carrying targeted amino acid substitutions. We show that His124 at an interface between two heterodimers of AGA is crucial for the thermodynamically stable oligomeric structure of AGA. Furthermore, the side chain of Thr206 is essential both for the proteolytic activation and enzymatic activity of AGA. Finally, the proper geometry of the residues His204-Asp205 seems to be crucial for the activation of AGA precursor polypeptides. We propose here a reaction mechanism for the activation of AGA which could be valid for homologous enzymes as well.     

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanasecleaving primarily cellobiose units from the non-reducing endof cellulose chains. The ß-l,4 glycosidic bond iscleaved by acid catalysis with an aspartic acid, D221, as thelikely proton donor, and another aspartate, D175, probably ensuringits protonation and stabilizing charged reaction intermediates.The catalytic base has not yet been identified experimentally.The refined crystal structure of CBHII also shows a tyrosineresidue, Y169, located close enough to the scissile bond tobe involved in catalysis. The role of this residue has beenstudied by introducing a mutation Y169F, and analysing the kineticand binding behaviour of the mutated CBHII. The crystal structureof the mutated enzyme was determined to 2.0 Å resolutionshowing no changes when compared with the structure of nativeCBHII. However, the association constants of the mutant enzymefor cellobiose and cellotriose are increased threefold and for4-methylumbelliferyl cellobioside over 50-fold. The catalyticconstants towards cellotriose and cellotetraose are four timeslower for the mutant. These data suggest that Y169, on interactingwith a glucose ring entering the second subsite in a narrowtunnel, helps to distort the glucose ring into a more reactiveconformation. In addition, a change in the pH activity profilewas observed. This indicates that Y169 may have asecond rolein the catalysis, namely to affect the protonation state ofthe active site carboxylates, D175 and D221.     

Probing the molecular basis of allergy. three-dimensional structure of the bovine lipocalin allergen Bos d 2

The three-dimensional structure of the major bovine allergen Bos d 2 has been determined by using x-ray diffraction at 1.8-A resolution. Structurally Bos d 2 is a member of the lipocalin family comprising proteins with transport functions. There is a flat small cavity inside the Bos d 2 protein core suitable for ligand binding, and it is possible that Glu115 and Asn37 inside the core are able to make hydrogen bonds with the ligand. Many allergens from different animals belong to the lipocalin family. The amino acid residue similarities between these lipocalins indicate putative regions for IgE binding. Comparison with the available allergen structures from other sources suggests that these allergens are roughly the same size and that their shape is more spherical than elliptical.     

Inversion of the roles of the nucleophile and acid/base catalysts in the covalent binding of epoxyalkyl xyloside inhibitor to the catalytic glutamates of endo-1,4-{beta}-xylanase (XYNII): a molecular dynamics study

X-Ray crystal structures have revealed that 2,3-epoxypropyl-ß-D-xylosidereacts with endo-1,4-ß-xylanase (XYNII) by forming a covalentbond with Glu86. In contrast, 3,4-epoxybutyl-ß-D-xylosideforms a covalent bond with Glu177. In the normal enzyme reactionGlu86 acts as the catalytic nucleophile and Glu177 as the acid/basecatalyst. To rationalize the observed reactivity of the twomechanism-based inhibitors, we carried out eight 300 ps moleculardynamics simulations for different enzyme–inhibitor complexes.Simulations were done for both stereo isomers (R and S) of theinhibitors and for enzyme in which the protonation state ofthe nucleophile and acid/base catalyst was normal (Glu86 charged,Glu177 neutral) and in which the roles of the catalytic residueswere reversed (Glu86 neutral, Glu177 charged). The number ofreactive conformations found in each simulation was used topredict the reactivity of epoxy inhibitors. The conformationwas considered to be a reactive one when at the same time (i)the proton of the catalytic acid was close (<2.9/3.4/3.9Å) to the oxirane oxygen of the inhibitor, (ii) the nucleophilewas close to the terminal carbon of the oxirane group (<3.4/3.9/4.4Å) and (iii) the nucleophile approached the terminal carbonfrom a reactive angle (<30/45/60° from an ideal attackangle). On the basis of the number of reactive conformations,2,3-epoxypropyl-ß-D-xyloside was predicted to form a covalentbond with Glu86 and 3,4-epoxybutyl-ß-D-xyloside with Glu177,both in agreement with the experiment. Thus, the MD simulationsand the X-ray structures indicate that in the covalent bindingof 3,4-epoxybutyl-ß-D-xyloside the roles of the catalyticglutamates of XYNII are reversed from that of the normal enzymereaction.     

Large-scale purification and preliminary x-ray diffraction studies of human aspartylglucosaminidase

Selective vestibular neurectomy is an effective treatment for intractable vertigo of peripheral vestibular origin when preservation of hearing is a goal. The retrolabyrinthine and retrosigmoid-intracanalicular approaches have been used predominantly at our institutions over the last 10 years. The results and complications of these two techniques were compared. No significant differences were found between hearing results in these two patient groups. The retrosigmoid-internal auditory can approach yielded better control of recurrent episodic vertigo, as well as superior ablation of postoperative ice-water caloric responses (p < 0.05). Surgical complications, including the incidence of cerebrospinal fluid leakage (greater in retrolabyrinthine approach) and postoperative headache (more prevalent in retrosigmoid approach), were also analyzed. To further evaluate the results of this study, data were reanalyzed and compared with previously published reports of selective vestibular nerve section.     

Comparison of numerical and computational aspects between two constraint-based contact methods in the description of wheel/rail contacts

Multibody System Dynamics - The numerical and computation aspects of the Knife-edge Equivalent Contact (KEC) constraint and lookup table (LUT) methods are compared in this paper. The LUT method...