Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target

Isoform IX of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), CA IX, is a transmembrane protein involved in solid tumor acidification through the HIF-1α activation cascade. CA IX has a very high catalytic activity for the hydration of carbon dioxide to bicarbonate and protons, even at acidic pH values (of around 6.5), typical of solid, hypoxic tumors, which are largely unresponsive to classical chemo- and radiotherapy. Thus, CA IX is used as a marker of tumor hypoxia and as a prognostic factor for many human cancers. CA IX is involved in tumorigenesis through many pathways, such as pH regulation and cell adhesion control. The X-ray structure of the catalytic domain of CA IX has been recently reported, being shown that CA IX has a typical α-CA fold. However, the CA IX structure differs significantly from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the proteoglycan (PG) domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. As all mammalian CAs, CA IX is inhibited by several main classes of inhibitors, such as the inorganic anions, the sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.), the phenols, and the coumarins. The mechanism of inhibition with all these classes of compounds is understood at the molecular level, but the sulfonamides and their congeners have important applications. It has been recently shown that both in vitro, in cell cultures, as well as in animals with transplanted tumors, CA IX inhibition with sulfonamides lead to a return of the extracellular pH to more normal values, which leads to a delay in tumor growth. As a consequence, CA IX represents a promising antitumor target for the development of anticancer agents with an alternative mechanism of action.     

Disulfide bond assignments of secreted Frizzled-related protein-1 provide insights about Frizzled homology and netrin modules.

Secreted Frizzled-related protein-1 (sFRP-1), a soluble protein that binds to Wnts and modulates Wnt signaling, contains an N-terminal domain homologous to the putative Wnt-binding site of Frizzled (Fz domain) and a C-terminal heparin-binding domain with weak homology to netrin. Both domains are cysteine-rich, having 10 and 6 cysteines in the Fz and heparin-binding domains, respectively. In this study, the disulfide linkages of recombinant sFRP-1 were determined. Numbering sFRP-1 cysteines sequentially from the N terminus, the five disulfide linkages in the Fz domain are 1-5, 2-4, 3-8, 6-10, and 7-9, consistent with the disulfide pattern determined for homologous domains of several other proteins. The disulfide linkages of the heparin-binding domain are 11-14, 12-15, and 13-16. This latter set of assignments provides experimental verification of one of the disulfide patterns proposed for netrin (NTR) modules and thereby supports the prediction that the C-terminal heparin-binding domain of sFRP-1 is an NTR-type domain. Interestingly, two subsets of sFRPs appear to have alternate disulfide linkage patterns compared with sFRP-1, one of which involves the loss of a disulfide due to deletion of a single cysteine from the NTR module, whereas the remaining cysteine may pair with a new cysteine introduced in the Fz domain of the protein. Analysis of glycosylation sites showed that sFRP-1 contains a relatively large carbohydrate moiety on Asn(172) (approximately 2.8 kDa), whereas Asn(262), the second potential N-linked glycosylation site, is not modified. No O-linked carbohydrate groups were detected. There was evidence of heterogeneous proteolytic processing at both the N and C termini of the recombinant protein. The predominant N terminus was Ser(31), although minor amounts of the protein with Asp(41) and Phe(50) as the N termini were observed. The major C-terminal processing event was removal of the terminal amino acid (Lys(313)) with only a trace amount of unprocessed protein detected.     

Structural characterization of recombinant soluble rat neuroligin 1: mapping of secondary structure and glycosylation by mass spectrometry

Neuroligins (NLs) are a family of transmembrane proteins that function in synapse formation and/or remodeling by interacting with beta-neurexins (beta-NXs) to form heterophilic cell adhesions. The large N-terminal extracellular domain of NLs, required for beta-NX interactions, has sequence homology to the alpha/beta hydrolase fold superfamily of proteins. By peptide mapping and mass spectrometric analysis of a soluble recombinant form of NL1, several structural features of the extracellular domain have been established. Of the nine cysteine residues in NL1, eight are shown to form intramolecular disulfide bonds. Disulfide pairings of Cys 117 to Cys 153 and Cys 342 to Cys 353 are consistent with disulfide linkages that are conserved among the family of alpha/beta hydrolase proteins. The disulfide bond between Cys 172 and Cys 181 occurs within a region of the protein encoded by an alternatively spliced exon. The disulfide pairing of Cys 512 and Cys 546 in NL1 yields a structural motif unique to the NLs, since these residues are highly conserved. The potential N-glycosylation sequons in NL1 at Asn 109, Asn 303, Asn 343, and Asn 547 are shown occupied by carbohydrate. An additional consensus sequence for N-glycosylation at Asn 662 is likely occupied. Analysis of N-linked oligosaccharide content by mass matching paradigms reveals significant microheterogeneous populations of complex glycosyl moieties. In addition, O-linked glycosylation is observed in the predicted stalk region of NL1, prior to the transmembrane spanning domain. From predictions based on sequence homology of NL1 to acetylcholinesterase and the molecular features of NL1 established from mass spectrometric analysis, a novel topology model for NL three-dimensional structure has been constructed.     

Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.     

猪瘟病毒石门强毒株和兔化弱毒疫苗株E2蛋白糖基化位点差异分析

猪瘟病毒强毒株和兔化弱毒疫苗株E2糖蛋白分别含有5个和6个潜在的糖基化位点,其中986N是兔化弱毒疫苗株所特有的。为了分析二者糖基化位点差异及其影响,将去掉信号肽和跨膜区的猪瘟病毒石门强毒株(Shi-men)和兔化弱毒疫苗株(HCLV)E2基因置于蜂素信号肽序列下游,使其在Sf9细胞内表达重组Shimen-E2和HCLV-E2蛋白。结果显示,重组E2蛋白以二聚体的形式分泌表达于细胞培养液中,但二者分子量存在差异。用endo H和PNGase F对纯化后的重组E2蛋白进行去糖基化处理后,二者分子量大小变成一致,证实石门强毒株和兔化弱毒株E2蛋白分子量大小的差异可能是由于糖基化程度的差异所致。对986N糖基化位点进行定点突变后发现,突变后的Shimen-E2与野生型HCLV-E2分子量大小一致,而突变后的HCLV-E2与野生型Shimen-E2分子量大小一致,表明Shimen-E2和HCLV-E2分子量大小的差异的确是由于986N糖基化位点的差异引起的。     

An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.     

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδ in vivo.     

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase

O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.     

Differential Glycosylation of Tractin and LeechCAM, Two Novel Ig Superfamily Members, Regulates Neurite Extension and Fascicle Formation

By immunoaffinity purification with the mAb Lan3-2, we have identified two novel Ig superfamily members, Tractin and LeechCAM. LeechCAM is an NCAM/FasII/ApCAM homologue, whereas Tractin is a cleaved protein with several unique features that include a PG/YG repeat domain that may be part of or interact with the extracellular matrix. Tractin and LeechCAM are widely expressed neural proteins that are differentially glycosylated in sets and subsets of peripheral sensory neurons that form specific fascicles in the central nervous system. In vivo antibody perturbation of the Lan3-2 glycoepitope demonstrates that it can selectively regulate extension of neurites and filopodia. Thus, these experiments provide evidence that differential glycosylation can confer functional diversity and specificity to widely expressed neural proteins.     

Expression and characterization of recombinant gamma-tryptase

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. gamma-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. gamma-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys(26) and Cys(145), that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed gamma-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd gamma-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc gamma-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of gamma-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that gamma-tryptase possesses a substrate preference and inhibitor profile distinct from that of beta-tryptase. Although the role of gamma-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of beta-tryptase.     

Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display

Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expected from expression levels and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing.     

Biochemical characterization of the thioredoxin domain of Escherichia coli DsbE protein reveals a weak reductant

Thioredoxin (Trx) domain is a typical fold functioning in thiol/disulfide exchange. DsbE protein is one of the Trx-domain containing proteins involved in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble C-terminal Trx domain of DsbE protein was overexpressed and purified to homogeneity. We herein report biochemical characterization of the structural and redox properties of this domain. During redox reaction, the domain undergoes a structural transformation resulting in a more stable reduced form with a free energy difference (DeltaDeltaG(Redox)) of ca. 5 kcal/mol, but the thiol/disulfide exchange exhibits very low reactivity. The standard redox potential (E0') for the active thiol/disulfide is -0.175 V and the pK(a) value of the active cysteine is around 6.8, indicating that the domain acts as a weak reductant. This implies that the membrane-anchored DsbE protein may provide driven reducing power for the redox reaction in the thiol/disulfide exchange pathway.     

Crystal structure of rice Rubisco and implications for activation induced by positive effectors NADPH and 6-phosphogluconate

The key enzyme of plant photosynthesis, D-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), must be activated to become catalytically competent via the carbamylation of Lys201 of the large subunit and subsequent stabilization by Mg(2+) coordination. Many biochemical studies have reported that reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 6-phosphogluconate (6PG) function as positive effectors to promote activation. However, the structural mechanism remains unknown. Here, we have determined the crystal structures of activated rice Rubisco in complex with NADPH, 6PG, or 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP). The structures of the NADPH and 6PG complexes adopt open-state conformations, in which loop 6 at the catalytic site and some other loops are disordered. The structure of the 2CABP complex is in a closed state, similar to the previous 2CABP-bound activated structures from other sources. The catalytic sites of the NADPH and 6PG complexes are fully activated, despite the fact that bicarbonate (NaHCO(3)) was not added into the crystallization solution. In the catalytic site, NADPH does not interact with Mg(2+) directly but interacts with Mg(2+)-coordinated water molecules, while 6PG interacts with Mg(2+) directly. These observations suggest that the two effectors promote Rubisco activation by stabilizing the complex of Mg(2+) and the carbamylated Lys201 with unique interactions and preventing its dissociation. The structure also reveals that the relaxed complex of the effectors (NADPH or 6PG), distinct from the tight-binding mode of 2CABP, would allow rapid exchange of the effectors in the catalytic sites by substrate D-ribulose 1,5-bisphosphate for catalysis in physiological conditions.     

The Venus's-flytrap and cysteine-rich domains of the human Ca2+ receptor are not linked by disulfide bonds

The extracellular N-terminal domain of the human Ca(2+) receptor (hCaR) consists of a Venus's-flytrap (VFT) domain and a cysteine-rich (Cys-rich) domain. We have shown earlier that the Cys-rich domain is critical for signal transmission from the VFT domain to the seven-transmembrane domain. The VFT domain contains 10 cysteines: two of them (Cys(129) and Cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (Cys(60)-Cys(101), Cys(358)-Cys(395), and Cys(437)-Cys(449)) are predicted to form three intramolecular disulfide bonds. The Cys-rich domain contains nine cysteines, the involvement of which in disulfide bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys(236) and Cys(482), form disulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site inserted between the VFT domain and the Cys-rich domain. These mutant hCaRs remain fully functional compared with the wild type hCaR. After TEV protease digestion of the mutant hCaR proteins, dimers of the VFT were identified on Western blot under nonreducing conditions. We concluded that there is no disulfide bond between the VFT and the Cys-rich domains in the hCaR.     

Glycosylation of skeletal calsequestrin: implications for its function

Calsequestrin (CASQ) serves as a major Ca(2+) storage/buffer protein in the sarcoplasmic reticulum (SR). When purified from skeletal muscle, CASQ1 is obtained in its glycosylated form. Here, we have confirmed the specific site and degree of glycosylation of native rabbit CASQ1 and have investigated its effect on critical properties of CASQ by comparison with the non-glycosylated recombinant form. Based on our comparative approach utilizing crystal structures, Ca(2+) binding capacities, analytical ultracentrifugation, and light-scattering profiles of the native and recombinant rabbit CASQ1, we propose a novel and dynamic role for glycosylation in CASQ. CASQ undergoes a unique degree of mannose trimming as it is trafficked from the proximal endoplasmic reticulum to the SR. The major glycoform of CASQ (GlcNAc(2)Man(9)) found in the proximal endoplasmic reticulum can severely hinder formation of the back-to-back interface, potentially preventing premature Ca(2+)-dependent polymerization of CASQ and ensuring its continuous mobility to the SR. Only trimmed glycans can stabilize both front-to-front and the back-to-back interfaces of CASQ through extensive hydrogen bonding and electrostatic interactions. Therefore, the mature glycoform of CASQ (GlcNAc(2)Man(1-4)) within the SR can be retained upon establishing a functional high capacity Ca(2+) binding polymer. In addition, based on the high resolution structures, we propose a molecular mechanism for the catecholaminergic polymorphic ventricular tachycardia (CPVT2) mutation, K206N.     

Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX

Posttranslational processing of vitamin K-dependent proteins includes gamma-carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acids. To determine whether carboxylation is directed by the propeptide sequence, homologous among the precursors of these proteins, alterations were made in the Factor IX propeptide cDNA. The extent of gamma-carboxylation of recombinant Factor IX was assessed using conformation-specific antibodies directed against the gamma-carboxyglutamic acid-dependent, metal-stabilized structure. Deletion of the propeptide (residues -18 to -1) abolished carboxylation, but not secretion, of Factor IX. Substitution of alanine for phenylalanine -16 or glutamic acid for alanine -10 also impaired carboxylation. These results indicate that the Factor IX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for gamma-carboxylation. The association of the propeptide with the gamma-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in posttranslational protein processing.