Single‐molecule pair studies of the interactions of the α‐GalNAc (Tn‐antigen) form of porcine submaxillary mucin with soybean agglutinin

Mucins form a group of heavily O‐glycosylated biologically important glycoproteins that are involved in a variety of biological functions, including modulating immune response, inflammation, and adhesion. Mucins are also involved in cancer and metastasis and often express diagnostic cancer antigens. Recently, a modified porcine submaxillary mucin (Tn‐PSM) containing GalNAcα1‐O‐Ser/Thr residues was shown to bind to soybean agglutinin (SBA) with ～10⁶‐fold enhanced affinity relative to GalNAcα1‐O‐Ser, the pancarcinoma carbohydrate antigen. In this study, dynamic force spectroscopy is used to investigate molecular pairs of SBA and Tn‐PSM. A number of force jumps that demonstrate unbinding or rebinding events were observed up to a distance equal to 2.0 μm, consistent with the length of the mucin chain. The unbinding force increased from 103 to 402 pN with increasing force loading rate. The position of the activation barrier in the energy landscape of the interaction was 0.1 nm. The lifetime of the SBA–TnPSM complex in the absence of applied force was determined to be in the range 1.3–1.9 s. Kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range 3.3 × 10^?3–2.5 × 10^?3 s. The long lifetime of the SBA‐TnPSM complex is compatible with a binding model in which lectin molecules “bind and jump” from α‐GalNAc residue to α‐GalNAc residue along the polypeptide chain of Tn‐PSM before dissociating. These findings have important implications for the molecular recognition properties of mucins. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 719–728, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Periostin is a collagen associated bone matrix protein regulated by parathyroid hormone

Periostin is a 90 kDa secreted protein, originally identified in murine osteoblast-like cells, with a distribution restricted to collagen-rich tissues and certain tumors. In this paper, we first analyzed the expression of periostin mRNA and protein in human fetal osteoblasts (hFOB) and human osteosarcoma (hOS) cell lines by RT real-time PCR and Western blot, respectively. The hFOB 1.19 and three hOS (MHM, KPDXM and Eggen) showed highly variable periostin mRNA levels and protein. Second, we showed that the expression of periostin mRNA was inversely related to the cells' abilities to differentiate and mineralize. Then, we investigated the regulation of periostin mRNA in hFOB after siRNA treatment and in mouse primary osteoblasts (mOB) treated with PTH. Knock-down of periostin mRNA, down-regulated PTHrP, but did not affect the expression of other important markers of differentiation such as RUNX2. In addition, periostin mRNA was transiently up-regulated in osteoblasts by PTH. Finally, the localization of periostin and its partially co-localization with collagen 1a1 mRNA and protein was studied in mouse embryos and postnatal pups using in situ hybridization and immunohistochemistry, respectively. In conclusion, the present study provides novel observations related to the expression, distribution and regulation of periostin in bone cells and extracellular matrix.     

Reduced VLDL clearance in Apoe−/−Npc1−/− mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe^−/−Npc1^−/− mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe^−/−Npc1^−/− liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe^−/−Npc1^−/− liver was unexpected. However, several other LXR target genes also increased in Apoe^−/−Npc1^−/− liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe^−/− mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe^−/−Npc1^−/− mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.     

Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C₅₀ carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C₄₀ lycopene, C₄₅ nonaflavuxanthin, C₅₀ flavuxanthin, and C₅₀ sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C₅₀ carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ɛ-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C₅₀ carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ɛ-cyclic C₅₀ carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ɛ- and γ-cyclic C₅₀ carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C₅₀ carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.Carotenoids are natural pigments synthesized by bacteria, fungi, algae, and plants, and more than 750 different carotenoids have been isolated from natural sources (17). They possess important biological functions as protectants against light and oxygen excess in photosynthetic processes (32, 38), and they have been proposed to reduce the risk of certain cancers, cardiovascular disease, and Alzheimer disease due to their antioxidative properties (20, 46). The global market for carotenoids used as food colorants and nutritional supplements was estimated at approximately $935 million in 2005 (11). More than 95% of all natural carotenoids are based on a symmetric C₄₀ phytoene backbone, and only a small number of C₃₀ and even fewer C₅₀ carotenoids have been discovered (42).C₅₀ carotenoids have multiple conjugated double bonds, and they contain at least one hydroxyl group; both these features contribute to strong antioxidative properties (17, 30, 32, 38). In nature, C₅₀ carotenoids are synthesized by bacteria of the order Actinomycetales, and to date, only two different C₅₀ carotenoid biosynthetic pathways have been described in the literature. The biosynthetic pathways of the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-ɛ,ɛ-carotene] and the β-cyclic C₅₀ carotenoid C.p.450 [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-β,β-carotene] have been elucidated in Corynebacterium glutamicum (22, 23) and in Dietzia sp. CQ4 (41), respectively. For both pathways, the common precursor, C₄₀ lycopene, is synthesized from C₁₅ farnesyl pyrophosphate (FPP) via the methylerythritol 4-phosphate (MEP) pathway, which is present in most eubacteria (33). Effective lycopene production has been achieved in genetically engineered noncarotenogenic hosts, such as Escherichia coli and Saccharomyces cerevisiae (9). Accordingly, the potential of using such biotechnologically relevant hosts for heterologous production of any lycopene-derived carotenoids has generated high interest.The biosynthesis of cyclic C₅₀ carotenoids from lycopene is catalyzed by lycopene elongase and carotenoid cyclases. Even though most carotenoids in plants and microorganisms exhibit cyclic structures, cyclization reactions were predominantly known for C₄₀ pathways (45) catalyzed by monomeric enzymes that have been isolated from plants and bacteria (5, 16, 27, 29, 31, 36). In C. glutamicum, the genes crtYe, crtYf, and crtEb were identified as being involved in the conversion of lycopene to the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin (22, 44). Sequential elongation of lycopene into the acyclic C₅₀ carotenoid flavuxanthin was catalyzed by the crtEb gene product lycopene elongase. Subsequent cyclization to decaprenoxanthin was catalyzed by a heterodimeric C₅₀ carotenoid, ɛ-cyclase, encoded by crtYe and crtYf (22). C. glutamicum can synthesize both mono- and diglucosylated decaprenoxanthin; however, the genetic and enzymatic bases for glucosylation of decaprenoxanthin are unknown. Analogous to decaprenoxanthin, biosynthesis of the β-cyclic C₅₀ carotenoid C.p.450 in Dietzia sp. CQ4 from lycopene involves lycopene elongase and C₅₀ carotenoid β-cyclase activities (41).While most cyclic carotenoids exhibit β-rings, ɛ-ring-containing pigments are common in higher plants (7), and carotenoids substituted only with γ-rings are rarely observed in plants and algae (14). To date, no biosynthetic pathway for γ-cyclic C₅₀ carotenoids has been reported in the literature.Micrococcus luteus NCTC2665 (the “Fleming strain”) is a Gram-positive bacterium belonging to the family Micrococcaceae within the order Actinomycetales. The carotenoids, including the γ-cyclic C₅₀ sarcinaxanthin [(2R,6R,2′R,6′R)-(2,2′-bis(4-hydroxy-3-methyl-2-butenyl)-γ,γ-carotene)], synthesized by this bacterium have been identified and structurally elucidated (26). We recently isolated and characterized several wild-type M. luteus strains from the sea surface microlayer of the middle part of the Norwegian coast (39). Here, we report one additional such marine M. luteus isolate, designated Otnes7, forming color-intensive colonies indicating high sarcinaxanthin production levels. Both Otnes7 and NCTC2665 were used as M. luteus model strains, and the sarcinaxanthin biosynthetic gene clusters were cloned from both strains. The complete sarcinaxanthin biosynthetic pathway from lycopene was elucidated, including glucosylation, and we also explored the potential of using Otnes7-derived genes to achieve effective heterologous production of sarcinaxanthin in E. coli. The results add important new knowledge of the biosynthesis of C₅₀ carotenoids, and in particular, they highlight the diverse functions of C₅₀ carotenoid cyclases leading to synthesis of structurally different carotenoids.     

Cold adapted features of Vibrio salmonicida catalase: characterisation and comparison to the mesophilic counterpart from Proteus mirabilis

The gene encoding catalase from the psychrophilic marine bacterium Vibrio salmonicida LFI1238 was identified, cloned and expressed in the catalase-deficient Escherichia coli UM2. Recombinant catalase from V. salmonicida (VSC) was purified to apparent homogeneity as a tetramer with a molecular mass of 235 kDa. VSC contained 67% heme b and 25% protoporphyrin IX. VSC was able to bind NADPH, react with cyanide and form compounds I and II as other monofunctional small subunit heme catalases. Amino acid sequence alignment of VSC and catalase from the mesophilic Proteus mirabilis (PMC) revealed 71% identity. As for cold adapted enzymes in general, VSC possessed a lower temperature optimum and higher catalytic efficiency (k _cat/K _m) compared to PMC. VSC have higher affinity for hydrogen peroxide (apparent K _m) at all temperatures. For VSC the turnover rate (k _cat) is slightly lower while the catalytic efficiency is slightly higher compared to PMC over the temperature range measured, except at 4°C. Moreover, the catalytic efficiency of VSC and PMC is almost temperature independent, except at 4°C where PMC has a twofold lower efficiency compared to VSC. This may indicate that VSC has evolved to maintain a high efficiency at low temperatures.     

Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose Biosynthesis

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.     

In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides

Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.     

WRN interacts physically and functionally with the recombination mediator protein RAD52

Werner syndrome (WS) is a premature aging disorder that predisposes affected individuals to cancer development. The affected gene, WRN, encodes an RecQ homologue whose precise biological function remains elusive. Altered DNA recombination is a hallmark of WS cells suggesting that WRN plays an important role in these pathways. Here we report a novel physical and functional interaction between WRN and the homologous recombination mediator protein RAD52. Fluorescence resonance energy transfer (FRET) analyses show that WRN and RAD52 form a complex in vivo that co-localizes in foci associated with arrested replication forks. Biochemical studies demonstrate that RAD52 both inhibits and enhances WRN helicase activity in a DNA structure-dependent manner, whereas WRN increases the efficiency of RAD52-mediated strand annealing between non-duplex DNA and homologous sequences contained within a double-stranded plasmid. These results suggest that coordinated WRN and RAD52 activities are involved in replication fork rescue after DNA damage.