Serum Differentially Modifies the Transcription and Translation of NMDAR Subunits in Retinal Neurons

The N-methyl-d-Aspartate type of glutamate receptor (NMDAR) plays a major role in the vertebrate retina. Expression of NR1 splice-variants and NR2 subunits in the retina differs from that in the brain, suggesting a tissue-specific heteromeric assembly of NMDARs. We previously demonstrated that serum alters retinal glutamate receptor properties. In order to relate this effect to NMDAR subunit composition, we here studied the effect of serum on the expression of NMDAR subunits and splice-variants in chick retinal neurons in primary culture. Our results show that mRNA and protein expression of NR1 alternative splice-variants and NR2 subunits are differentially modified by glutamate contained in serum. Such alteration suggests that NMDAR structure is reversed to embryonic heteromeric composition, through the control of subunit availability. The present findings could be relevant for the understanding of the lack of effect in the retina, of drugs which have been shown to protect cortical neurons from glutamate-induced excitotoxicity in those pathological or clinical conditions in which the retina is exposed to serum. Special issue article in honor of Dr. Ricardo Tapia.     

Structures of vertebrate hyaluronidases and their unique enzymatic mechanism of hydrolysis

Human hyaluronidases (Hyals) are a group of five endo-beta-acetyl-hexosaminidase enzymes, Hyal-1, -2, -3, -4, and PH-20, which degrade hyaluronan using a hydrolytic mechanism of action. Catalysis by these Hyals has been shown to follow a double-displacement scheme. This involves a single Glu residue within the enzyme, the only catalytic residue, as the proton donor (acid). Also involved is a carbonyl group of the hyaluronan (HA) N-acetyl-D-glucosamine as a unique type of nucleophile. Thus the substrate participates in the mechanism of action of its own catalysis. An oxocarbonium ion transition state is postulated, but there is no formation of a covalent enzyme-glycan intermediate, as found in most such reactions. The major domain is catalytic and has a distorted (beta/alpha)8 triose phosphate isomerase (TIM) barrel fold. The C-terminal domain is separated by a peptide linker. Each Hyal has a different C-terminal sequence and structure, the function of which is unknown. These unique C-termini may participate in the additional function(s) associated with these multifunctional enzymes.     

Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains

The structure of Janus kinases (JAKs) is unique among protein tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patients with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specifically, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of cells with these mutants demonstrated that, although they were constitutively phosphorylated basally, they were unable to transmit cytokine-dependent signals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or its deletion from the full-length molecule reduced catalytic activity. Through coimmunoprecipitation of the isolated pseudokinase domain with the isolated catalytic domain, we provide the first evidence that these two domains interact. Furthermore, whereas the wild-type pseudokinase domain modestly inhibited kinase domain-mediated STAT5 phosphorylation, the patient-derived mutants markedly inhibited this phosphorylation. We thus conclude that the JAK3 pseudokinase domain is essential for JAK3 function by regulating its catalytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that increased inhibition of kinase activity by the pseudokinase domain likely contributes to the disease pathogenesis in these two patients.     

Genome-based identification of a carbohydrate binding module in Streptococcus pneumoniae hyaluronate lyase

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the connective tissues of higher animals, thereby destroying the normal connective tissue structure and exposing the host tissue cells to various endo- and exogenous factors, including bacterial toxins. The 3D crystal structures of functionally active but truncated Streptococcus pneumoniae and S. agalactiae hyaluronate lyases, along with their substrate and product complexes, have been determined. The enzymes are multidomain proteins with helical barrel-like catalytic domains and two types of beta-sheet domains. Here, through genome-based bioinformatics studies we identify an additional beta-sheet domain present in the most N-terminal part of streptococcal hyaluronate lyases. Fold recognition and modeling studies show that the domain is structurally similar to carbohydrate binding modules and is therefore likely to be directly involved in hyaluronan binding. Likely carbohydrate binding residues were identified and electrostatic complementarity of the hyaluronate lyase domain with hyaluronan demonstrated. The newly identified presumed hyaluronan binding domain likely improves catalytic efficiency by colocalizing the enzyme and its substrate. Other possible functions are discussed. Two contacting aromatic residues are conserved in the hydrophobic core of the hyaluronate lyase domain and in many, perhaps all, families in the superfamily in which they may be placed. This observation may help the identification and classification of other carbohydrate binding modules.     

Link protein has greater affinity for versican than aggrecan

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.     

Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.     

Organization of the human PTK7 gene encoding a receptor protein tyrosine kinase-like molecule and alternative splicing of its mRNA

Protein tyrosine kinase-7 (PTK7) is a receptor protein tyrosine kinase (RPTK)-like molecule that contains a catalytically inactive tyrosine kinase domain. We report here the genomic structure of the human PTK7 gene by screening a BAC library and DNA sequencing. The PTK7 gene is organized into 20 exons. All of the splicing junctions followed the conserved GT/AG rule. The exon-intron structure of the PTK7 gene in the region that encodes the catalytic domain was distinct from those of other RPTKs with strong homology. The 5'-flanking sequence of the PTK7 gene contains two GC boxes that concatenate Sp1 binding motifs, but does not contain either the TATA or CAAT consensus sequence. Using a luciferase reporter assay, it was demonstrated that the 883-bp 5'-flanking sequence is functional as a promoter of the PTK7 gene. We identified four new splicing variants in testis that could be derived from alternative splicing of exons 8-10, 10, a part of 12-13, and 16. The expression patterns of the splicing variants in the hepatoma and colon cancer cells were different from those of the testis. Our findings suggest that PTK7 is evolutionarily distinct from other RPTKs, and that the alternative splicing of PTK7 mRNA may contribute to its diverse function in cell signaling.     

Insights into the mechanism of action of hyaluronate lyase: role of C-terminal domain and Ca2+ in the functional regulation of enzyme

Hyaluronate lyases (HLs) cleave hyaluronan and certain other chondroitin/chondroitin sulfates. Although native HL from Streptococcus agalactiae is composed of four domains, it finally stabilizes after autocatalytic conversion as a 92-kDa enzyme composed of the N-terminal spacer, middle alpha-, and C-terminal domains. These three domains are independent folding/unfolding units of the enzyme. Comparative structural and functional studies using the enzyme and its various fragments/domains suggest a relatively insignificant role of the N-terminal spacer domain in the 92-kDa enzyme. Functional studies demonstrate that the alpha-domain is the catalytic domain. However, independently it has a maximum of only about 10% of the activity of the 92-kDa enzyme, whereas its complex with the C-terminal domain in vitro shows a significant enhancement (about 6-fold) in the activity. It has been previously proposed that the C-terminal domain modulates the enzymatic activity of HLs. In addition, one of the possible roles for calcium ions was suggested to induce conformational changes in the enzyme loops, making HL more suitable for catalysis. However, we observed that calcium ions do not interact with the enzyme, and its role actually is in modulating the hyaluronan conformation and not in the functional regulation of enzyme.     

Potential molecular targeting of splice variants for cancer treatment

Array of new targets for investigation as cancer therapeutics has great potential to grow as new splice-variants are identified and characterized in cancer cell-lines and tumor samples. Tumor-specific splice variants are being discovered at an increasing rate and their functions are also investigated in cancer progression. The tumor-specific splice variants whose expression patterns and activities are successfully characterized may become attractive targets for ablation or splicing modification. The extreme specificity of their expression suggests that a variant-specific treatment may allow for targeting of cancerous cells with minimal impact to healthy tissues. Clinical investigation of applying antisense oligonucleotides to down-regulate mRNAs that contribute to cancer cell survival and to modify splicing patterns in muscular dystrophy has shown promising results. These results show that antisense therapy may be applied effectively and safely in humans. As these treatment strategies continue to improve and novel tumor-specific splice-variants are identified, modification of splicing patterns will become an important field of investigation to develop more effective and safe cancer therapies.     

Hyaluronidase can modulate expression of CD44

CD44 is a family of transmembrane glycoproteins with multiple isoforms generated by alternative exon splicing of a single gene. CD44 and its variants are expressed on a wide variety of cells including cancer cells. The mechanisms by which splice variant exons are selected are unknown. The presence of hyaluronan in the environment of the cell appears to influence that selection process. The expression of particular splice variants of CD44 as well as the simultaneous presence of hyaluronan is important for motility, invasion, and the metastatic spread of some tumors. The influence of hyaluronidase digestion on the expression of CD44 in human cancer cell lines was examined. CD44 isoforms containing alternatively spliced exons were sensitive to hyaluronidase digestion in all lines examined, but differences between cell lines were observed. Expression of CD44s, the standard form, was resistant to digestion in two of three cell lines. A tentative model was formulated proposing that CD44 isoforms containing splice variants are unstable, requiring the continuous presence of ligand for expression. CD44s is relatively more stable, not requiring the continuous presence of hyaluronan. Additionally, a number of new CD44 variant isoforms, not previously observed, were identified.     

Evidence for the Heparin-Binding Ability of the Ascidian Xlink Domain and Insight into the Evolution of the Xlink Domain in Chordates

The vertebrate Xlink domain is found in two types of genes: lecticans and their associated hyaluronan-and-proteoglycan-binding-link-proteins (HAPLNs), which are components of the extracellular matrix, and those represented by CD44 and stabilins, which are expressed on the surface of lymphocytes. In both types of genes, Xlink functions as a hyaluronan binding domain. We have already reported that protochordate ascidians possess only the latter type of gene. The present analysis of the expression of ascidian Xlink domain genes revealed that these genes function in blood cell migration and apoptosis. While the Xlink domain is found in various metazoans, including ascidians and nematodes, hyaluronan is believed to be specific for vertebrates. In comprehensive genome surveys for hyaluronan synthase (HAS), we found no HAS gene in ascidians. We also established that hyaluronan is absent from the ascidian body biochemically. Therefore, ascidians possess the Xlink domain, but they lack HA. We recovered one ascidian Xlink domain gene that encoded a heparin-binding protein, although it shows no affinity for hyaluronan. Based on these findings, we conclude that in invertebrates, the Xlink domain serves as heparin-binding protein domain and functions in blood cell migration and apoptosis. Its binding affinity for HA might have been acquired in the vertebrate lineage.     

Identification of an alternative form of caspase-9 in human gastric cancer cell lines: a role of a caspase-9 variant in apoptosis resistance

Overcoming apoptosis resistance to chemotherapy and radiation may lead to a reduction in gastric cancer death. We hypothesize that the apoptotic machinery in gastric cancer cells is dependent upon specific cellular conditions. In the course of our study of the expression of apoptosis-related genes in human gastric cancer cell lines, we have identified a cDNA clone which predicts an alternative form of caspase-9. The caspase-9 variant, which we designated as caspase-9 beta, retained a truncated structure of native caspase-9 without its catalytic domain and was expressed in seven cell lines from human gastric cancer. Among the cell lines examined, MKN-28 cells, which exhibited the most resistance against apoptotic stimuli, expressed the highest level of caspase-9 beta. The induction of apoptosis by staurosporine or actinomycin D was markedly suppressed in caspase-9 beta-transfected HeLa cells. These results are consistent with our hypothesis that the caspase-9 beta may be an endogenous dominant-negative molecule which attenuates apoptotic activity in human gastric cancer cells.     

High-resolution structure of a modular hyperthermostable endo-β-1,4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain

The endo-β-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-β-1,4-mannanase structure determined. The structure exhibits two domains, a (β/α)₈-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like β-sandwich fold formed of seven β-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.     

RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability

The receptor for hyaluronan-mediated motility (RHAMM), an acidic coiled coil protein, has previously been characterized as a cell surface receptor for hyaluronan, and a microtubule-associated intracellular hyaluronan binding protein. In this study, we demonstrate that a subset of cellular RHAMM localizes to the centrosome and functions in the maintenance of spindle integrity. We confirm a previous study showing that the amino terminus of RHAMM interacts with microtubules and further demonstrate that a separate carboxy-terminal domain is required for centrosomal targeting. This motif overlaps the defined hyaluronan binding domain and bears 72% identity to the dynein interaction domain of Xklp2. RHAMM antibodies coimmunprecipitate dynein IC from Xenopus and HeLa extracts. Deregulation of RHAMM expression inhibits mitotic progression and affects spindle architecture. Structure, localization, and function, along with phylogenetic analysis, suggests that RHAMM may be a new member of the TACC family. Thus, we demonstrate a novel centrosomal localization and mitotic spindle-stabilizing function for RHAMM. Moreover, we provide a potential mechanism for this function in that RHAMM may cross-link centrosomal microtubules, through a direct interaction with microtubules and an association with dynein.     

Kinetic properties of Streptococcus pneumoniae hyaluronate lyase

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment. Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of K(m)s for all of these different enzymes.