2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation.

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of 'misfire' products of Rubisco.

Control of periplasmic interdomain thiol:disulfide exchange in the transmembrane oxidoreductase DsbD.

The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys(103)-Cys(109) disulfide of nDsbD by Cys(461) of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously ( Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370, 643-658 ) that the attacking cysteine (Cys(461)) in isolated cDsbD has a high pK(a) value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK(a) values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK(a) values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK(a) values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.

Order within a mosaic distribution of mitochondrial c-type cytochrome biogenesis systems?

Mitochondrial cytochromes c and c(1) are present in all eukaryotes that use oxygen as the terminal electron acceptor in the respiratory chain. Maturation of c-type cytochromes requires covalent attachment of the heme cofactor to the protein, and there are at least five distinct biogenesis systems that catalyze this post-translational modification in different organisms and organelles. In this study, we use biochemical data, comparative genomic and structural bioinformatics investigations to provide a holistic view of mitochondrial c-type cytochrome biogenesis and its evolution. There are three pathways for mitochondrial c-type cytochrome maturation, only one of which is present in prokaryotes. We analyze the evolutionary distribution of these biogenesis systems, which include the Ccm system (System I) and the enzyme heme lyase (System III). We conclude that heme lyase evolved once and, in many lineages, replaced the multicomponent Ccm system (present in the proto-mitochondrial endosymbiont), probably as a consequence of lateral gene transfer. We find no evidence of a System III precursor in prokaryotes, and argue that System III is incompatible with multi-heme cytochromes common to bacteria, but absent from eukaryotes. The evolution of the eukaryotic-specific protein heme lyase is strikingly unusual, given that this protein provides a function (thioether bond formation) that is also ubiquitous in prokaryotes. The absence of any known c-type cytochrome biogenesis system from the sequenced genomes of various trypanosome species indicates the presence of a third distinct mitochondrial pathway. Interestingly, this system attaches heme to mitochondrial cytochromes c that contain only one cysteine residue, rather than the usual two, within the heme-binding motif. The isolation of single-cysteine-containing mitochondrial cytochromes c from free-living kinetoplastids, Euglena and the marine flagellate Diplonema papillatum suggests that this unique form of heme attachment is restricted to, but conserved throughout, the protist phylum Euglenozoa.

Localization of the delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity.

The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wild-type epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications.

Cellular and molecular studies of Marfan syndrome mutations identify co-operative protein folding in the cbEGF12-13 region of fibrillin-1.

Human fibrillin-1 is an extra-cellular matrix glycoprotein with a modular organisation that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats interspersed with transforming growth factor beta binding protein-like (TB) domains. We have studied Marfan syndrome-causing mutations which affect calcium binding to cbEGF13, and demonstrate that in human fibroblast cells they cause unexpected endoplasmic reticulum retention, indicative of a folding defect. Biochemical and biophysical studies of in vitro refolded fragments from the TB3-cbEGF14 region indicate long-range and unidirectional effects of these substitutions on the adjacent N-terminal domain cbEGF12. In contrast, only short-range effects of a pathogenic mutation affecting calcium binding to cbEGF19 are observed, and secretion of this mutant protein occurs. Further NMR studies on wild-type cbEGF12-13 and cbEGF12-14 identify a co-operative dependence of domain folding where calcium binding to cbEGF13 is required before cbEGF12 can adopt a native Ca(2+)-dependent fold. These data demonstrate that during biosynthesis of fibrillin-1, multiple tandem repeats of cbEGF domains may not necessarily fold independently and therefore missense mutations resulting in identical substitutions may have different effects on the fate of the mutant protein. Complex folding of modular proteins should therefore be considered when interpreting the molecular pathology of single-gene disorders.

How tyrosine 15 phosphorylation inhibits the activity of cyclin-dependent kinase 2-cyclin A.

Inhibition of cyclin-dependent kinase 1 (CDK1) activity by Tyr-15 phosphorylation directly regulates entry into mitosis and is an important element in the control of the unperturbed cell cycle. Active site phosphorylation of other members of the CDK family that regulate cell cycle progression instates checkpoints that are fundamental to eukaryotic cell cycle regulation. Kinetic and crystallographic analyses of CDK2-cyclin A complexes reveal that this inhibitory mechanism operates through steric blockade of peptide substrate binding and through the creation of an environment that favors a non-productive conformation of the terminal group of ATP. By contrast, tyrosine phosphorylation of CDK2 alters neither its Km for ATP nor its significant intrinsic ATPase activity. Tyr-15-phosphorylated CDK2 retains trace protein phosphorylation activity that should be considered in quantitative and qualitative cell cycle models.

Precise identification of a human immunodeficiency virus type 1 antigen processing mutant.

Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences.

Interaction of mannan binding lectin with alpha2 macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.

The catalytically active serine protease domain of human complement factor I.

Factor I (fI) is a major regulator of complement. As a protease it has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH). Cleavage of C3b by fI yields iC3b, a major opsonin. The cleavage occurs through the formation of a ternary complex between the enzyme, the substrate, and the cofactor. The catalytic subunit of fI, the SP domain, accommodates substrate recognition and cleavage. The role of the fI heavy chain within the catalysis complex is unknown. Using partial proteolysis and affinity chromatography an intact form of the SP domain was generated and isolated from fI in high yield. fI and the SP domain were found to have similar amidolytic activities but strikingly different proteolytic activities on C3(NH(3)). fI did not cleave C3(NH(3)) in the absence of fH, while in its presence it cleaved C3(NH(3)) rapidly at two sites. The SP domain, however, slowly cleaved C3(NH(3)) in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. Pefabloc SC and antipain inhibited the proteolytic activity of both fI and the SP domain, but suramin inhibited only fI and not the SP domain. The contrast in the proteolytic activities suggests that the heavy chain domains and the cofactor must have roles in orienting the natural substrates and restricting cleavage to the two sites which yield iC3b through a highly specific catalysis.

Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan.

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.

Identification and removal of O-linked and non-covalently linked sugars from recombinant protein produced using Pichia pastoris.

The use of the methylotrophic yeast Pichia pastoris for large-scale recombinant production of proteins for therapeutic uses and/or biophysical characterisation has been gaining popularity. Here we describe the use of this organism for the production of a von Willebrand factor C domain from procollagen IIA for solution NMR studies. In this research, we specifically identified sites of O-linked glycosylation on the expressed protein, although the native protein is not glycosylated. We demonstrated that it was possible to remove the oligosaccharides by enzymatic digestion, however this approach proved to be prohibitively expensive for the scale of production required for high-resolution structural studies by NMR spectroscopy. After removal of the O-linked glycosylation sites by site-directed mutagenesis, we confirmed that the protein was no longer covalently glycosylated. However, analysis by 1H- and 13C-edited spectroscopy identified the presence of non-covalently associated glycans which were removed by lectin affinity chromatography. We have synthesised methods for the identification and removal of both covalently and non-covalently bound oligosaccharides from heterologous protein expressed in P. pastoris.

Structural consequences of cysteine substitutions C1977Y and C1977R in calcium-binding epidermal growth factor-like domain 30 of human fibrillin-1.

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.

Gamma III-crystallin is the primary target of glycation in the bovine lens incubated under physiological conditions.

Several mechanisms have been proposed for the way in which glucose and its metabolites cause cataract, retinopathy and other complications of diabetes, the most convincing being glycation. Glycation, the reaction of sugars with free amino groups of proteins, is one of a variety of non-enzymic post-translational modifications. The aim of the present study was to identify some of the most reactive proteins in the lens when incubated under physiological conditions. Fresh intact bovine lenses were incubated with [14C]glucose in a conventional tissue-culture medium with added antibiotics. After 3 and 6 days of incubation, the water-soluble proteins were separated by size-exclusion chromatography. Glycated proteins from the water-soluble fractions were separated by using a sugar affinity column (Affi-Gel 601). Then the radioactive fractions were identified on SDS/polyacrylamide gels. In addition, the whole bovine lenses were incubated with 10 mM fructose and glucose for 3 and 6 days. The glycated proteins from the water-soluble fractions in parallel with the radioactive fractions were separated by affinity chromatography, and were identified further by amino-acid sequencing. A progressive uptake of radioactive label showed that the majority of proteins incorporating both glucose and fructose were water-soluble fractions. Chromatography and SDS/polyacrylamide gel results showed that alpha- and gamma-crystallin and some proteins of a mean molecular mass of 36-37 kDa incorporated sugars early during incubation. After 6 days of incubation, more crystallins were glycated compared with 3 days, in particular beta-crystallin. Affinity-chromatography results indicated that proteins with subunit masses of 36 kDa and 20 kDa were possibly radiolabelled at an early stage. The purified glycated proteins following incubation with both glucose and fructose, which corresponded to 20 kDa and 36 kDa bands on SDS/polyacrylamide gels, were sequenced by Edman degradation. N-terminal sequences of both 20 kDa bands were Gly-Lys-Ile-Thr, characteristic of gamma-crystallins, but the N-termini of both 36 kDa bands were blocked. Further sequencing after digestion of 36 kDa bands with trypsin and running on HPLC revealed that the glucose sample gave the peptide sequences as Gly-Glu-Tyr-Pro-Asp-Tyr-Gln-Gln and Tyr-Glu-Leu-Pro-Asn-Tyr-Arg, which match with bovine gammaIIIb-crystallin. The peptide sequence Tyr-Glu-Leu-Pro-Asn-Tyr-Arg is only present in the published sequence of bovine gammaIIIb-crystallin and not in any other type of gamma-crystallin. The fructose sample gave the peptide sequences Ile-Thr-Phe-Tyr-Glu-Asp-Arg, Arg-Gly-Asp-Tyr-Pro-Asp-Tyr-Gln-Gln-Trp, Gln-Tyr-Leu-Leu-Arg and Val-Val-Asp-Leu-Tyr, which all matched with bovine gammaIIIa-crystallin. The sequence Val-Val-Asp-Leu-Tyr only appears in the sequence of bovine gammaIIIa-crystallin. gammaIII-Crystallin is the most susceptible lens protein to glycation. The primary target of glucose is gammaIIIb-crystallin, whereas that of fructose is gammaIIIa-crystallin. The early glycation of gammaIII-crystallin by glucose and fructose could result in structural alterations, leading to aggregation of crystallin and eventually cataract formation.

Identification and characterization of a novel interaction between pulmonary surfactant protein D and decorin.

Surfactant-associated protein D (SP-D) is a collectin that is present in lung surfactant and mucosal surfaces. Although SP-D regulates diverse functions, only a few proteins are known to bind to this collectin. Here we describe the co-purification of decorin, a novel SP-D-binding protein, from amniotic fluid. The human decorin that co-purified with SP-D is a 130-150-kDa proteoglycan, which has a 46-kDa protein core and approximately 90-kDa dermatan sulfate chain. Both native and recombinant decorin can bind to SP-D that is already bound to maltose-agarose matrix, and these SP-D-decorin complexes are dissociated at high salt (0.5-1.0 m NaCl) conditions, releasing the decorin. We further show that SP-D and decorin interact with each other (kd = 4 nm) by two mechanisms. First, the direct binding and competition experiments show that the carbohydrate recognition domain (CRD) of SP-D binds in a calcium dependent-manner to the sulfated N-acetyl galactosamine moiety of the glycosaminoglycan chain. Second, complement component C1q, a complement protein that is known to interact with decorin core protein via its collagen-like region, partially blocks the interaction between decorin and native SP-D. This protein, however, does not block the interaction between decorin and SP-D(n/CRD), a recombinant fragment that lacks the N-terminal and collagen-like regions. Furthermore, the core protein, obtained by chondroitin ABC lyase treatment of decorin, binds SP-D, but not SP-D(n/CRD). These findings suggest that decorin core protein binds the collagen-like region of the SP-D. Concentrations of decorin and SP-D are negatively correlated to each other, in amniotic fluid, implying a functional relevance for SP-D-decorin interaction, in vivo. Collectively, our results show that carbohydrate recognition domains of SP-D interact with the dermatan sulfate moiety of decorin via lectin activity and that the core protein of decorin binds the collagen-like region of SP-D in vitro, and these interactions may be operative in vivo.

Cancer-associated cleavage of cytokeratin 8/18 heterotypic complexes exposes a neoepitope in human adenocarcinomas.

The intermediate filament network in simple glandular epithelial cells predominantly consists of heterotypic complexes of cytokeratin 8 (K8) and cytokeratin 18 (K18). In contrast to other cytokeratins, K8 and K18 are persistently expressed during malignant transformation, but changes in cell morphology are accompanied by alterations in the intermediate filament network. To study molecular changes, K8 and K18 were purified from surgically removed colon cancer and normal epithelia tissues. Western blotting and amino acid sequencing revealed the presence of abundant K8 and K18 fragments, truncated at the N terminus, from cancerous, but not normal, epithelial cells. The fragmentation pattern indicates proteolysis mediated by several enzymes, including trypsin-like enzymes. The cancer-associated forms of K8 and K18 are specifically recognized by the human antibody, COU-1, cloned from the B cells of a cancer patient. We demonstrate that COU-1 recognizes a unique conformational epitope presented only by a complex between K8 and K18. The epitope is revealed after proteolytic removal of the head domain of either K8 or K18. A large panel of recombinant K8 and K18 fragments, deleted N- or C-terminally, allowed for the localization of the COU-1 epitope to the N-terminal part of the rod domains. Using surface plasmon resonance, the affinity of COU-1 for this epitope was determined to be 10(9) x m(-1), i.e. more than 2 orders of magnitude higher than for intact heterotypic K8/K18 complexes. The cellular distribution of truncated K8/K18 heterotypic complexes in viable adenocarcinomas cells was probed using COU-1 showing small fibrillar structures distinct from those of intact K8/K18 complexes. Previously we demonstrated the binding and subsequent internalization of recombinant Fab COU-1 to live cancer cells. We have thus characterized a cancer neoepitope recognized by the humoral immune system. The results have biological as well as clinical implications.