Ligand Blot Analysis Research Articles

Function of CTLGA9 Amino Acid Residue Leucine-6 in Modulating Cry Toxicity.

Aedes aegypti is a crucial vector for many arboviral diseases that cause millions of deaths worldwide and thus is of major public health concern. Crystal (Cry) proteins, which are toxins produced by Bacillus thuringiensis, are structurally organized into three-domains, of which domain II is the most variable in terms of binding towards various toxin receptors. The binding of Cry11Aa to putative receptor such as aminopeptidase-N (APN) is explicitly inhibited by midgut C-type lectins (CTLs). The similarity between the domain II fold of Cry11Aa toxin and the carbohydrate recognition domain in the CTLs is a possible structural basis for the involvement of Cry domain II in the recognition of carbohydrates on toxin receptors. In this study, a site-directed point mutation was introduced into the A. aegypti CTLGA9 gene on the basis of molecular docking findings, leading to substitution of the Leucine-6 (Leu-6) residue in the protein with alanine. Subsequently, functional monitoring of the mutated protein was carried out. Unlike the amino acid residues of wild-type CTLGA9, none of the residues of mutant (m) CTLGA9 were competed with Cry11Aa for binding to the APN receptor interface. Additionally, ligand blot analysis showed that both wild-type and mutant CTLGA9 had similar abilities to bind to APN and Cry11Aa. Furthermore, in the competitive ELISA in which labeled mutant CTLGA9 (10 nM) was mixed with increasing concentrations of unlabeled Cry11Aa (0–500 nM), the mutant showed no competition with Cry11Aa for binding to APN., By contrast, in the positive control sample of labeled wild type CTLGA9 mixed with same concentrations of Cry11Aa competition between the two ligands for binding to the APN was evident. These results suggest that Leucine-6 may be the key site involved in the competitive receptor binding between CTLGA9 and Cry11Aa. Moreover, according to the bioassay results, mutant CTLGA9 could in fact enhance the toxicity of Cry11Aa. Our novel findings provide further insights into the mechanism of Cry toxicity as well as a theoretical basis for enhancing the mosquitocidal activity of these toxin through molecular modification strategies.

Triosephosphate isomerase of Streptococcus pneumoniae is released extracellularly by autolysis and binds to host plasminogen to promote its activation.

Recruitment of plasminogen is an important infection strategy of the human pathogen Streptococcus pneumoniae to invade host tissues. In Streptococcus aureus, triosephosphate isomerase (TPI) has been reported to bind plasminogen. In this study, the TPI of S. pneumoniae (TpiA) was identified through proteomic analysis of bronchoalveolar lavage fluid from a murine pneumococcal pneumonia model. The binding kinetics of recombinant pneumococcal TpiA with plasminogen were characterized using surface plasmon resonance (SPR, Biacore), ligand blot analyses, and enzyme‐linked immunosorbent assay. Enhanced plasminogen activation and subsequent degradation by plasmin were also shown. Release of TpiA into the culture medium was observed to be dependent on autolysin. These findings suggest that S. pneumoniae releases TpiA via autolysis, which then binds to plasminogen and promotes its activation, thereby contributing to tissue invasion via degradation of the extracellular matrix.

Inhibition of Bemisia tabaci vectored, GroEL mediated transmission of tomato leaf curl New Delhi virus by garlic leaf lectin (Allium sativum leaf agglutinin)

GroEL or symbionin synthesized by the endosymbionts of whitefly (Bemisia tabaci)/ aphids play a cardinal role in the persistent, circulative transmission of plant viruses by binding to viral coat protein/ read-through protein. Allium sativum leaf agglutinin (ASAL), a Galanthus nivalis agglutinin (GNA)- related mannose-binding lectin from garlic leaf has been reported as a potent controlling agent against hemipteran insects including whitefly and aphids. GroEL related chaperonin- symbionin was previously identified as a receptor of ASAL by the present group in the brush border membrane vesicle (BBMV) of mustard aphid. In the present study similar GroEL receptor of ASAL has been identified through LC-MS/MS in the BBMV of B. tabaci which serves as a vector for several plant viruses including tomato leaf curl New Delhi virus (ToLCNDV). Ligand blot analysis of ASAL-fed B. tabaci showed that when GroEL is pre-occupied by ASAL, it completely blocks its further binding to ToLCNDV coat protein (ToLCNDV-CP). Prior feeding of ASAL hindered the co-localization of ToLCNDV-CP and GroEL in the midgut of B. tabaci. Immunoprecipitation followed by western blot with ASAL-fed B. tabaci yielded similar result. Moreover, ASAL feeding inhibited viral transmission by B. tabaci. Together, these results confirmed that the interaction of ASAL with GroEL interferes with the binding of ToLCNDV-CP and inhibits further B. tabaci mediated viral transmission.

Insulin-Like Growth Factor Binding Protein-3 Binds to Histone 3.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is an essential protein that regulates cellular processes such as cell proliferation, apoptosis, and differentiation. It is known to bind with several proteins to carry out various cellular functions. In this study, we report for the first time that IGFBP-3 is a histone 3 (H3) binding protein. Sub-cellular fractionation was performed to separate into cytosolic fraction, nucleic acid binding protein fraction and insoluble nuclear fraction. Using ligand blot analysis, we identified a ~15 kDa protein that can interact with IGFBP-3 in the insoluble nuclear fraction. The 15 kDa protein was confirmed as histone 3 by far-Western blot analysis and co-immunoprecipitation experiments. A dot-blot experiment further validated the binding of IGFBP-3 with H3. The intensity of IGFBP-3 on dot-blot showed a proportional increase with H3 concentrations between 2.33 pmol–37.42 pmol. Our results support the presence of protein-protein interaction between IGFBP-3 and H3. The physical binding between IGFBP-3 and H3 could indicate its yet another cellular role in regulating the chromatin remodeling for gene transcription.

Detection of Fibronectin-Binding Proteins of Streptococcus pyogenes Using Ligand Blot Analysis.

Streptococcus pyogenes utilizes extracellular cellular matrix (ECM) proteins to adhere to human tissues and internalize into host cells. Fibronectin (Fn) is one of the most abundant ECM proteins and targeted by a wide variety of secreted Fn-binding proteins (Fbps) of S. pyogenes. However, prior to detailed kinetic analysis of that binding process, evaluations of the ability of S. pyogenes strains to bind to Fn as well as interactions of target molecules with Fn are required. In this chapter, we present routine procedures for ligand blot analysis with labeled human Fn, using bacterial cell wall extracts prepared by either enzymatic digestion of cells or extraction with a denaturing agent.

Investigation on the difference of insecticidal activities between Vip3Aa11 and Vip3Aa39 proteins against Helicoverpa armigera (Insecta: Lepidoptera)

Vip3Aa11 and Vip3Aa39 were isolated from Bacillus thuringiensis strains C9 and T03B001, respectively. Despite that there is 95.06% similarity of amino acid sequences between these two proteins, the insecticidal activity of them against Helicoverpa armigera is different and their LC50 were 35 µg/ml and 120 µg/ml, respectively. In order to explore the causes of differences in the insecticidal activity of these two proteins, we have examined the trypsin sensitivity assay and the assay binding with the brush border membrane vesicles of Helicoverpa armigera. The experiment of treatment with trypsin indicated that there is no difference in sensitivity between these two proteins, as both proteins were able to bind to the midgut BBMV of Helicoverpa armigera. However, the binding ability of Vip3Aa11 was stronger than that of Vip3Aa39. The ligand-blotting analysis demonstrated both biotinylated Vip3Aa11 and Vip3Aa39 toxins primarily showed binding to four midgut putative receptor proteins of 65, 80, 100 and 120 kDa. These results provide that the causes for the different insecticidal activity between these two proteins against Helicoverpa armigera may be due to the difference in binding ability on BBMV.

Transcytosis of Junonia coenia densovirus VP4 across the gut epithelium of Spodoptera frugiperda (Lepidoptera: Noctuidae).

The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells. The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel, rather than in the gut. In this study, we used fall armyworm, Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two-dimensional ligand blot analysis. We also assessed the rate of flux of the primary viral structural protein, VP4 fused to eGFP with a proline-rich linker (VP4-P-eGFP) through the gut epithelium ex vivo in an Ussing chamber. The mechanisms involved with transcytosis of VP4-P-eGFP were assessed by use of inhibitors. Bovine serum albumin (BSA) and eGFP were used as positive and negative control proteins, respectively. In contrast to BSA, which binds to multiple proteins on the brush border membrane, VP4-P-eGFP binding was specific to a protein of high molecular mass. Protein flux was significantly higher for VP4-P-eGFP after 2 h than for albumin or eGFP, with rapid transcytosis of VP4-P-eGFP within the first 30 min. In contrast to BSA which transcytosed following clathrin-mediated endocytosis, the movement of VP4-P-eGFP was vesicle-mediated but clathrin-independent. The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S. frugiperda.

Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests

To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.

Expression of glyceraldehyde-3-phosphate dehydrogenase on the surface of Clostridium perfringens cells

During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C. perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin. Although GAPDH has no signal sequence, it is expressed on the cell surface of many microorganisms. The presence of GAPDH on the surface of C. perfringens cells was analyzed using ELISA and flow cytometry analyses; purified rGAPDH bound to the surface of C. perfringens cells. As autolysin is reportedly involved in the binding of GAPDH to the cell surface, we evaluated the interaction between rGAPDH and the C. perfringens autolysin Acp by both ELISA and ligand blotting assay. These assays revealed that rGAPDH binds to the catalytic domain of Acp but not the cell wall binding domains. These results suggest that autolysin mediates expression of GAPDH on the surface of C. perfringens cells and indicate a possible moonlighting function for GAPDH in binding both Fn and plasminogen.

Surfactant protein A down-regulates epidermal growth factor receptor by mechanisms different from those of surfactant protein D

We recently reported that the lectin surfactant protein D (SP-D) suppresses epidermal growth factor receptor (EGFR) signaling by interfering with ligand binding to EGFR through an interaction between the carbohydrate-recognition domain (CRD) of SP-D and N-glycans of EGFR. Here, we report that surfactant protein A (SP-A) also suppresses EGF signaling in A549 human lung adenocarcinoma cells and in CHOK1 cells stably expressing human EGFR and that SP-A inhibits the proliferation and motility of the A549 cells. Results with 125I-EGF indicated that SP-A interferes with EGF binding to EGFR, and a ligand blot analysis suggested that SP-A binds EGFR in A549 cells. We also found that SP-A directly binds the recombinant extracellular domain of EGFR (soluble EGFR or sEGFR), and this binding, unlike that of SP-D, was not blocked by EDTA, excess mannose, or peptide:N-glycosidase F treatment. We prepared a collagenase-resistant fragment (CRF) of SP-A, consisting of CRD plus the neck domain of SP-A, and observed that CRF directly binds sEGFR but does not suppress EGF-induced phosphorylation of EGFR in or proliferation of A549 cells. These results indicated that SP-A binds EGFR and down-regulates EGF signaling by inhibiting ligand binding to EGFR as well as SP-D. However, unlike for SP-D, SP-A lectin activity and EGFR N-glycans were not involved in the interaction between SP-A and EGFR. Furthermore, our results suggested that oligomerization of SP-A is necessary to suppress the effects of SP-A on EGF signaling.

Sensing Mg2+ contributes to the resistance of Pseudomonas aeruginosa to complement-mediated opsonophagocytosis.

Pseudomonas aeruginosa adaptation to survive in the host hinges on its ability to probe the environment and respond appropriately. Rapid adaptation is often mediated by two-component regulatory systems, such as the PhoP/PhoQ system that responds to Mg2+ ion concentration. However, there is limited information about the role of PhoQ in P. aeruginosa bloodstream infections. We used a murine model of systemic infection to test the virulence of a PhoQ-deficient mutant. Mutation of PhoQ impaired the virulence and the ability to cause bacteremia of P. aeruginosa. In the presence of blood concentrations of Mg2+ , a PhoQ mutant bound more C3 and was more susceptible to complement-mediated opsonophagocytosis than the parent strain, suggesting a direct effect of the Mg2+ on the modulation of expression of a bacterial component controlled by the PhoP/PhoQ system. Ligand blot analysis, C3 binding experiments and opsonophagocytosis assays identified this component as the outer membrane protein OprH, expression of which impaired the virulence of P. aeruginosa in a murine model of systemic infection. We demonstrate that expression of PhoQ is essential to detect Mg2+ and reduce the expression of OprH, a previously unrecognized C3 binding molecule that promotes the opsonophagocytosis of P. aeruginosa.

Transgenic Bt rice lines producing Cry1Ac, Cry2Aa or Cry1Ca have no detrimental effects on Brown Planthopper and Pond Wolf Spider

Transgenic rice expressing cry genes from the bacterium Bacillus thuringiensis (Bt rice) is highly resistant to lepidopteran pests. The brown planthopper (BPH, Nilaparvata lugens) is the main non-target sap-sucking insect pest of Bt transgenic rice. The pond wolf spider (PWS, Pardosa pseudoannulata) is one of the most dominant predators of BPH in rice fields. Consequently, the safety evaluation of Bt rice on BPH and PWS should be conducted before commercialization. In the current study, two experiments were performed to assess the potential ecological effects of Bt rice on BPH and PWS: (1) a tritrophic experiment to evaluate the transmission of Cry1Ac, Cry2Aa and Cry1Ca protein in the food chain; and (2) binding assays of Cry1Ac, Cry2Aa and Cry1Ca to midgut brush border membrane proteins from BPH and PWS. Trace amounts of the three Cry proteins were detected in BPH feeding on Bt rice cultivars, but only Cry1Ac and Cry2Aa proteins could be transferred to PWS through feeding on BPH. In vitro binding of biotinylated Cry proteins and competition assays in midgut protein vesicles showed weak binding, and ligand blot analysis confirmed the binding specificity. Thus, we inferred that the tested Bt rice varieties have negligible effects on BPH and PWS.

Membrane androgen receptor characteristics of human ZIP9 (SLC39A) zinc transporter in prostate cancer cells: Androgen-specific activation and involvement of an inhibitory G protein in zinc and MAP kinase signaling

Characteristics of novel human membrane androgen receptor (mAR), ZIP9 (SLC39A9), were investigated in ZIP9-transfected PC-3 cells (PC3-ZIP9). Ligand blot analysis showed plasma membrane [3H]-T binding corresponds to the position of ZIP9 on Western blots which suggests ZIP9 can bind [3H]-T alone, without a protein partner. Progesterone antagonized testosterone actions, blocking increases in zinc, Erk phosphorylation and apoptosis, further evidence that ZIP9 is specifically activated by androgens. Pre-treatment with GTPγS and pertussis toxin decreased plasma membrane [3H]-T binding and blocked testosterone-induced increases in Erk phosphorylation and intracellular zinc, indicating ZIP9 is coupled to an inhibitory G protein (Gi) that mediates both MAP kinase and zinc signaling. Testosterone treatment of nuclei and mitochondria which express ZIP9 decreased their zinc contents, suggesting ZIP9 also regulates free zinc through releasing it from these intracellular organelles. The results show ZIP9 is a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells.

Detection of a specific pattern of hyaluronan oligosaccharides and their binding proteins in human ovarian tumour.

Tumour cells generate hyaluronan (HA) oligomers (O-HA) by an autocrine mechanism to regulate their own behaviour through receptor interaction, necessitating analysis of HA sizes and its receptor expression in tumour progression. In this study for the first time, we identified specific size of HA in malignant ovarian tumour compared to benign tumour tissue. Therefore, we prepared the identified HA probes and conducted multiplex and monoplex ligand blot analysis and Immunohistochemistry to identify their receptor expression and distribution. Although, HA recognized CD44 as principle receptors despite of size, multiplex analysis showed multiple receptor expression with distribution at the tumour cell surface. Furthermore, the HA 6-mer (major O-HA of ovarian tumour) pull down of tumour tissue proteins showed 120 kDa protein along with CD44 with over expression in the malignant tumour. Upon depletion of CD44 protein HA 6-mer showed a major 120 kDa protein with distribution at nuclear membrane, suggesting that this protein may play an important role in ovarian tumour progression. In summary, ovarian tumour cells of different grade showed heterogeneity in generation of HA oligomers and their interaction with specific receptors. Therefore, simultaneous analysis of O-HA and their receptors expression could serve as a prognostic indicator during tumorigenesis. Copyright © 2016 John Wiley & Sons, Ltd. Down regulation of hyaluronan (HA) at tumour epithelial cells results in the generation of O-HA. Increasing evidence about O-HA biological functions (mainly in vitro) are available, but we lack information of O-HA sizes generated during tumorigenesis and less information is available about their receptor interaction. We used biochemical approaches and identified that tumour cells of different grade possessing heterogeneity in generation of O-HA and specificity towards receptor binding. We identified a new receptor for HA 6-mer that over express in cancer tissue that shows its role in tumour progression. Collectively, simultaneous identification of HA sizes and their receptors could serve as a prognostic marker.

Identification of an alkaline phosphatase as a putative Cry1Ac binding protein in Ostrinia furnacalis (Guenée)

Asian corn borer (ACB), Ostrinia furnacalis, is an important insect pest of maize susceptible to different Cry1A toxins. Based on amino acid sequence alignment of ALP sequences from lepidopteran larvae an alp gene was cloned from ACB, named ofalp. Pull dawn assays using biotinylated Cry1Ac and brush border membrane vesicles isolated from second instar ACB larvae showed that four proteins of 50, 65, 68 and 70kDa precipitated with the Cry1Ac. The 65kDa band cross-reacted with the anti-OfALP monoclonal antibody. GalNac was able to release the binding of Cry1Ac to the 65kDa OfALP in pull down assays. A 37kDa fragment from residues D173 to D473 of OfALP was cloned and expressed in Escherichia coli cells. We show that this ALP-fragment was able to bind Cry1Ac in ligand blot analysis. Our data also indicate that different ALP isoforms or variants may be also Cry1Ac binding proteins since more ALP enzymatic activity was pull down with Cry1Ac than with anti-OfALP antibody. We also analyzed the expression levels of ALP throughout the larval development by qPCR and ALP enzymatic activity. Our data indicated that ALP expression in ACB was observed preferentially in young instar larvae. Finally, we show that resistance in O. furnacalis ACB-AcR strain resistant to Cry1Ac did not correlate with changes in expression of this ALP protein since it shows similar gene expression of ofalp than the susceptible insect strain. Identification of Cry1Ac receptors will help to understand mechanism of action of Cry1Ac in O. furnacalis and to understand mechanism of Cry toxin resistance. Our data indicate that at least one ALP protein is involved in the binding interaction with Cry1Ac in O. furnacalis.

Inhibin Biosynthesis and Activity Are Limited by a Prodomain-Derived Peptide.

Gonadal-derived inhibin A and B are essential factors in mammalian reproduction, negatively regulating pituitary production of FSH. Inhibins are synthesized as heterodimers of α- and β-subunits, each comprising an N-terminal pro- and C-terminal mature domain. After dimerization, the inhibin α- and β-subunit prodomains are enzymatically cleaved from the mature domains at consensus RXXR sites (site1). Interestingly, the inhibin α-subunit is a unique TGF-β ligand, comprising a second cleavage site (site2) within its prodomain. Cleavage at site2 in the inhibin α-subunit prodomain releases a 43-amino acid proα-peptide. We aimed to determine the influence of the proα-peptide on inhibin synthesis and bioactivity. Blocking proα-peptide release by silencing cleavage site2 (Arg56-Arg61) inhibited both inhibin A and B synthesis. Ligand blot analysis and solid-phase binding assays indicated that the proα-peptide binds specifically to a mature 30-kDa inhibin (mean Kd 86 nM) but was unable to bind related activins. The proα-peptide suppressed inhibin A and B bioactivity in primary rat pituitary cell cultures. Mechanistically, the proα-peptide blocked inhibin A binding to its coreceptor, betaglycan (IC50 131 nM), and the subsequent sequestration of the activin type II receptor (IC50 156 nM), which underscores inhibin's biological activity. Based on the sequential mutations across the inhibin α-subunit, the proα-peptide binding site was localized to residues Arg341-Thr354, corresponding directly to the betaglycan binding region. Together our findings indicate that the proα-peptide limits the synthesis and bioactivity of inhibins.

A deletion mutant ndv200 of the Bacillus thuringiensis vip3BR insecticidal toxin gene is a prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insect damage.

Ectopic expression of a deletion mutant ( ndv200 ) of Bacillus thuringiensis vip3BR gene in tobacco plant provided almost complete protection against major crop pests cotton boll worm ( Helicoverpa armigera ), black cut worm ( Agrotis ipsilon ) and cotton leaf worm ( Spodoptera littoralis ). Whereas vip3BR transgenic tobacco plant failed to protect themselves from these insects and showed resistance towards cotton leaf worm only. An analogous form of the Bacillus thuringiensis vip3Aa insecticidal toxin gene, named vip3BR, was identified and characterized, and exhibited similar attributes to the well-known Vip3Aa toxin. Vip3BR possessed broad-spectrum lepidopteran-specific insecticidal properties effective against most major crop pests of the Indian subcontinent. A Vip3BR toxin protein N-terminal deletion mutant, Ndv200, showed increased insecticidal potency relative to the native toxin, which conferred efficacy against four major crop pests, including cotton boll worm (Helicoverpa armigera), black cut worm (Agrotis ipsilon), cotton leaf worm (Spodoptera littoralis), and rice yellow stem borer (Scirpophaga incertulas). Ligand blot analysis indicated the Ndv200 toxin recognized the same larval midgut receptors as the native Vip3BR toxin, but differed from receptors recognized by Cry1A toxins. In the present study, we tested the prospect of the vip3BR and ndv200 toxin gene as candidate in development of insect-resistant genetically engineered crop plants by generating transgenic tobacco plant. The study revealed that the ndv200 mutant of vip3BR insecticidal toxin gene is a strong and prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insects.

Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows

Hormones and metabolites act as satiety signals in the brain and play an important role in the control of feed intake (FI). These signals can reach the hypothalamus and brainstem, 2 major centers of FI regulation, via the blood stream or the cerebrospinal fluid (CSF). During the early lactation period of high-yielding dairy cows, the increase of FI is often insufficient. Recently, it has been demonstrated that insulin-like growth factors (IGF) may control FI. Thus, we asked in the present study if IGF-binding proteins (IGFBP) are regulated during the periparturient period and in response to feed restriction and therefore might affect FI as well. In addition, we specifically addressed conditional distribution of IGFBP in plasma and CSF. In one experiment, 10 multiparous German Holstein dairy cows were fed ad libitum and samples of CSF and plasma were obtained before morning feeding on d −20, −10, +1, +10, +20, and +40 relative to calving. In a second experiment, 7 cows in second mid-lactation were sampled for CSF and plasma after ad libitum feeding and again after feeding 50% of the previous ad libitum intake for 4 d. Intact IGFBP-2, IGFBP-3, and IGFBP-4 were detected in plasma by quantitative Western ligand blot analysis. In CSF, we were able to predominantly identify intact IGFBP-2 and a specific IGFBP-2 fragment containing detectable binding affinities for biotinylated IGF-II. Whereas plasma concentrations of IGFBP-2 and IGFBP-4 increased during the periparturient period, IGFBP-3 was unaffected over time. In CSF, concentrations of IGFBP-2, both intact and fragmented, were not affected during the periparturient period. Plasma IGF-I continuously decreased until calving but remained at a lower concentration in early lactation than in late pregnancy. Food restriction did not affect concentrations of IGF components present in plasma or CSF. We could show that the IGFBP profiles in plasma and CSF are clearly distinct and that changes in IGFBP in plasma do not simply correspond in the brain. We thus assume independent control of IGFBP distribution between plasma and CSF. Due to the known anorexic effect of IGF-I, elevated plasma concentrations of IGFBP-2 and IGFBP-4 during the postpartum period in conjunction with reduced plasma IGF-I concentrations may be interpreted as an endocrine response against negative energy balance in early lactation in dairy cows.

A Novel Pex14 Protein-interacting Site of Human Pex5 Is Critical for Matrix Protein Import into Peroxisomes

Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57-71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar α-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner.

Biochemical characterization of cathepsin D from the mussel Lamellidens corrianus

A lysosomal cathepsin D (EC 3.4.23.5) was purified to homogeneity from the soft tissues of the fresh water mussel (Lamellidens corrianus) by pepstatin A affinity chromatography. The purified enzyme is a glycoprotein and migrates as a single protein species in native PAGE and shows a single band in SDS-PAGE corresponding to a molecular mass of ~43 kDa. Under both these conditions cathepsin D hydrolyzes hemoglobin as shown by zymogram analysis. The purified enzyme cross-reacts with an antiserum to purified starfish (Asterias rubens) cathepsin D. Additionally, the enzyme was recognized by the starfish lysosomal enzyme targeting receptors (mannose 6-phosphate receptors: MPR 300 and 46) in ligand blot analysis. The KM value of the purified enzyme with hemoglobin is 1.5mM. pH and temperature optimum for the enzyme are 3.5 and 60 °C respectively.