Molecular pathogenesis of a disease: structural consequences of aspartylglucosaminuria mutations.

A deficiency of functional aspartylglucosaminidase (AGA) causes a lysosomal storage disease, aspartylglucosaminuria (AGU). The recessively inherited disease is enriched in the Finnish population, where 98% of AGU alleles contain one founder mutation, AGU(Fin). Elsewhere in the world, we and others have described 18 different sporadic AGU mutations. Many of these are predicted to interfere with the complex intracellular maturation and processing of the AGA polypeptide. Proper initial folding of AGA in the endoplasmic reticulum (ER) is dependent on intramolecular disulfide bridge formation and dimerization of two precursor polypeptides. The subsequent activation of AGA occurs autocatalytically in the ER and the protein is transported via the Golgi to the lysosomal compartment using the mannose-6-phosphate receptor pathway. Here we use the three-dimensional structure of AGA to predict structural consequences of AGU mutations, including six novel mutations, and make an effort to characterize every known disease mutation by dissecting the effect of mutations on intracellular stability, maturation, transport and the activity of AGA. Most mutations are substitutions replacing the original amino acid with a bulkier residue. Mutations of the dimer interface prevent dimerization in the ER, whereas active site mutations not only destroy the activity but also affect maturation of the precursor. Depending on their effects on the AGA polypeptide the mutations can be categorized as mild, moderate or severe. These data contribute to the expanding body of knowledge pertaining to molecular pathogenesis of AGU.

X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization.

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

Structural comparison of Ntn-hydrolases.

The Ntn-hydrolases (N-terminal nucleophile) are a superfamily of diverse enzymes that has recently been characterized. All of the proteins in this family are activated autocatalytically; they contain an N-terminally located catalytic nucleophile, and they cleave an amide bond. In the present study, the structures of four enzymes of this superfamily are compared in more detail. Although the amino acid sequence homology is almost completely absent, the enzymes share a similar alphabeta betaalpha-core structure. The central beta-sheets in the core were found to have different packing angles, ranging from 5 to 35 degrees. In the Ntn-hydrolases under study, eight totally conserved secondary structure units were found (region C). Five of them were observed to contain the greatest number of conserved and functionally important residues and are therefore crucial for the structure and function of Ntn-hydrolases. Two additional regions, consisting of secondary structure units (regions A and B), were found to be in structurally similar locations, but in different orders in the polypeptide chain. The catalytic machinery is located in the structures in a similar manner, and thus the catalytic mechanisms of all of the enzymes are probably similar. However, the substrate binding and the oxyanion hole differed partially.

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.

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.

Primary folding of aspartylglucosaminidase. Significance of disulfide bridges and evidence of early multimerization.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme involved in the degradation of N-linked glycoproteins in lysosomes. AGA is synthesized as an inactive precursor molecule, which is rapidly activated in the endoplasmic reticulum by a proteolytic cleavage into alpha- and beta-subunits. We have recently determined the three-dimensional structure of AGA and shown that it is a globular molecule with a heterotetrameric (alphabeta)2 structure. On the basis of structural and functional analyses, AGA seems to be the first mammalian protein belonging to a newly described protein family, the N-terminal nucleophile hydrolases. Because the activation of the prokaryotic members of the N-terminal nucleophile hydrolase family seems to be triggered by the assembly of the subunits, we have studied the initial folding and oligomerization of AGA and provide evidence that dimerization of two precursor molecules in the endoplasmic reticulum is a prerequisite for the activation of AGA. To gain further information on the structural determinants influencing the early folding of AGA, we used site-specific mutagenesis of cysteine residues to define the role of intrachain disulfide bridges in the folding and activation of the enzyme. The N-terminal disulfide bridges in both the alpha- and beta-subunits seem to have only a stabilizing role, whereas the C-terminal disulfide bridge in both subunits evidently plays an important role in the early folding and activation of AGA.

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

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes part in the ordered degradation of glycoproteins and a deficiency of which results in a lysosomal accumulation disease aspartylglucosaminuria in human. The mature enzyme consists of 24-kDa and 17-kDa subunits, which are both heterogeneously glycosylated. Activation of the enzyme from a single precursor polypeptide into two subunits is accomplished in the endoplasmic reticulum (ER). The relative lack of this proteolytic capacity in several tested high-producing expression systems has complicated the production of active recombinant enzyme in high quantities, which would be an alternative for purification of this molecule for crystallization. Consequently, the AGA enzyme has to be purified directly from cellular or tissue sources for crystallographic analysis. Here we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enzyme represents a heterogeneous pool of molecules not only due to glycosylation, but also heterogeneity at the polypeptide level, as demonstrated here. We were able to isolate a homogeneous peptide pool that was successfully crystallized and preliminary X-ray data collected from the crystals. The crystals diffract well to 2.0 angstroms and are thus suitable for determination of the crystal structure of AGA.

Three-dimensional structure of human lysosomal aspartylglucosaminidase.

The high resolution crystal structure of human lysosomal aspartylglucosaminidase (AGA) has been determined. This lysosomal enzyme is synthesized as a single polypeptide precursor, which is immediately post-translationally cleaved into alpha- and beta-subunits. Two alpha- and beta-chains are found to pack together forming the final heterotetrameric structure. The catalytically essential residue, the N-terminal threonine of the beta-chain is situated in the deep pocket of the funnel-shaped active site. On the basis of the structure of the enzyme-product complex we present a catalytic mechanism for this lysosomal enzyme with an exceptionally high pH optimum. The three-dimensional structure also allows the prediction of the structural consequences of human mutations resulting in aspartylglucosaminuria (AGU), a lysosomal storage disease.

A peptide C-terminal to the second Zn finger of human vitamin D receptor is able to specify nuclear localization.

A peptide of 27 amino acids, VDR(102-76), representing residues 76-102 immediately C-terminal to the second Zn finger of human vitamin D receptor (hVDR) was conjugated to fluorescein-labelled IgG using a bifunctional coupling reagent, m-maleimidobenzoyl n-hydroxysuccinimide. Upon microinjection into the cytoplasm of human osteosarcoma MG-63 cells, the chimeras accumulated in the nuclei. This transport was arrested by chilling or energy depletion. Two other peptides, VDR(80-67), spanning the N-terminal part of VDR(102-76), and VDR(108-97), spanning the C-terminal part of VDR(102-76), were not able to target the linked proteins to the nuclei. SV40(135-112), a peptide containing a well-characterized nuclear localization sequence (amino acids 112-135) of simian virus 40 (SV40) large T-antigen, caused complete nuclear accumulation under the same conditions. Wheat germ agglutinin, which inhibits SV40(135-112) transport, also inhibited the nuclear accumulation of VDR(102-76) as did energy depletion.