Quantification of cytokines involved in wound healing using surface plasmon resonance

Sensing of three cytokines related to chronic wound healing, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), with detection limits at or below 1 ng/mL in buffered saline solution and spiked cell culture medium (CCM) has been achieved. Fiber-optic surface plasmon resonance (SPR) sensors are coated with an antibody binding layer and antibodies specific to the cytokine of interest are covalently attached to this layer. To achieve such detection limits in a complex medium such as CCM, total protein content of 4 mg/mL, the use of a novel N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid (NHS-MHA) is necessary. A comparison of the detection limits for IL-6 using currently widely used CM-dextran and NHS-MHA shows an improvement by a factor of 3 using NHS-MHA. The detection limits for the monitoring of cytokines in spiked saline solutions and CCM were similar for TNF-alpha and slightly higher for IL-1 and IL-6. The detection of each cytokine in the presence of interfering agents resulted in concentration prediction well within the error of calibration. The SPR sensors are stable in CCM after 20 min of pretreatment in CCM, minimizing the reliance on a reference sensor to quantify the cytokines in complex media. This technique enables a major advancement in the field of real-time monitoring of biologically relevant molecules in complex biological fluids.     

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex

Molecular recognition and site-directed mutagenesis are usedin combination to identify kinetically, transition state interactionsbetween glucoamylase (GA) and the substrate maltose. Earlierstudies of mutant Glu180 – Gin GA had indicated a rolein substrate binding for Glul80 (Slerks, M.R., Ford, C., Reilly,P.J. and Svensson, B. (1990) Protein Engng, 3, 193–198).Here, changes in activation energies calculated from measuredk_cat/K_m values for a series of deoxygenated maltose analoguesindicate hydrogen bonding between the mutant enzyme and the3-OH group of the reducing end sugar ring. Using the same substrateanalogues and determining activation energies with wild-typeGA an additional hydrogen bond with the 2-OH group of maltoseis attributed to an interaction with the carboxylate Glu180.This novel combination of molecular recognition and site-directedmutagenesis enables an enzyme substrate transition state contactto be identified and characterized even without access to thethree dimensional structure of the enzyme. Given the distantstructural relationships between glucoamylases and several starchhydrolases (Svensson, B. (1988) FEBS Lett., 230, 72–76),such identified contacts may ultimately guide tailoring of theactivity of these related enzymes.     

Protein engineering of the relative specificity of glucoamylase from Aspergillus awamori based on sequence similarities between starch-degrading enzymes

Aspergillus glucoamylase catalyzes hydrolysis of D-glucose fromnon-reducing ends of starch with an {small tilde}300-fold {k^JKm) preference for the a-1,4- over the a-l,6-glucosidic linkagedetermined using the substrates maltose and iso-maltose. Itis postulated that as most amylolytic enzymes act on eitherthe a-1,4- or a-l,6-linkages, sequence comparison between active-siteregions should enable the correlation of the substrate bondspecificity with particular residues at key positions. Therefore,the already high bond-type selectivity in Aspergillus glucoamylasecould theoretically be augmented further by three single mutations,Serll9 Tyr, Glyl83 Lys and Serl84 His, in two separate active-siteregions. These mutants all had slight increases in activityas compared with the wild-type enzyme towards the a-l,4-linkedmaltose; this was due to lower Km values as well as small decreasesin activity towards isomaltose. This latter decrease in activitywas a result of higher Km values and a decrease in fc^, forthe Serl84 His mutant As a consequence, the selectivity of thethree glucoamylase mutants for a-1,4- over a-l,6-linked disaccharidesis enhanced 2.3- to 3.5-fold. In addition, the introductionof a cationic side chain in Glyl83 Lys and Serl84 His glucoamylase,broadens the optimal pH range for activity towards acidic aswell as alkaline conditions.     

Site-directed mutagenesis at the active site Trpl20 of Aspergillus awamori glucoamylase

Trpl20 of Aspergillus awamori glucoamylase has previously beenshown by chemical modification to be essential for activityand tentatively to be located near subsite 4 of the active site.To further test its role, restriction sites were inserted inthe cloned A.awamori gene around the Trpl20 coding region, andcassette mutagenesis was used to replace it with His, Leu, Pheand Tyr. All four mutants displayed 2% or less of the maximalactivity (k_cat) of wild-type glucoamylase towards maltose andmaltoheptaose. MichaelLs constants (K_M) of mutants decreased2- to 3-fold for maltose and were essentially unchanged formaltoheptaose compared with the wild type, except for a >3-fold decrease for maltoheptaose with the Trp120 – Tyrmutant. This mutant also bound isomaltose more strongly andhad more selectivity for its hydrolysis than wild-type glucoamylase.A subsite map generated from malto-oligosaecharide substrateshaving 2 – 7 D-glucosyl residues indicated that subsites1 and 2 had greater affinity for D-glucosyl residues in theTrp120 – Tyr mutant than in wild-type glucoamylase. Theseresults suggest that Trpl20 from a distant subsite is crucialfor the stabilization of the transition-state complex in subsites1 and 2.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies

Isolation of antibodies to antigens that are either unstable, exist in multiple morphologies or have very limited availability can be prohibitively difficult. Here we describe a novel technique combining the capabilities of phage display antibody technology and atomic force microscopy (AFM) that is used to isolate antibody fragments that bind to a specific morphology of the target antigen, alpha-synuclein. AFM imaging allows us to both visualize the presence and morphology of the target antigen as well as to monitor the efficiency of each step in the bio-panning process. We demonstrate that phage displayed antibodies specific to the target antigen morphology can be isolated after only two rounds of selection. The target antigen, alpha-synuclein, has been correlated with the Parkinson's disease (PD). Accumulation of alpha-synuclein fibrillar aggregates into Lewy body inclusions is a hallmark feature of PD. While alpha-synuclein can form several different aggregate morphologies including oligomers, protofibrils and fibrils, the role of these morphologies in the progression of PD is not known. The successful selection of the recombinant antibody described here can have potential therapeutic value since the single-chain fragment variable (scFv) can be expressed intracellularly to control folding and toxicity of the specific protein aggregates.     

Solvent and viscosity effects on the rate-limiting product release step of glucoamylase during maltose hydrolysis

The effects of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7), a novel neuroprotective compound, on the voltage-sensitive sodium channels (VSSC) were examined in the rat brain and cardiac myocytes. NS-7 inhibited [3H]batrachotoxinin A 20 alpha-benzoate (BTX) binding (neurotoxin receptor site 2) in brain membranes with a Ki value of 1 microM, while the compound was less effective in the cardiac myocytes (Ki = 13 microM). Aconitine, on the other hand, inhibited [3H]BTX binding to brain membranes and cardiac myocytes with the same potency. In contrast. NS-7 had no affinity for [3H]saxitoxin binding in brain (neurotoxin receptor site 1). In superfused slices of the rat cerebral cortex, NS-7 inhibited the veratridine (5 microM)-evoked glutamate release in a concentration-dependent manner, the IC50 value of which was 7.7 microM, whereas the compound showed a weak and not significant suppression of KCl-evoked glutamate release. The tissue concentrations of NS-7 in the rat cerebral cortex and heart were 89 and 28 nmole/g tissue, respectively, 5 min after its intravenous injection (8 mg/kg). Furthermore, in the cerebral cortex, NS-7 distributed preferentially to the membrane-enriched synaptosomal fraction. Since neurotoxin receptor site 2 is located in the transmembrane region of the VSSC moiety, the channel function may be substantially inhibited by a peripheral administration of NS-7. These results suggest that the blockade of neurotoxin receptor site 2 of VSSC in the brain contributes to the neuroprotective action of NS-7.     

Identification of enzyme-substrate and enzyme-product complexes in the catalytic mechanism of glucoamylase from Aspergillus awamori

Intermediates in the catalytic mechanism of Aspergillus awamori glucoamylase (GA) were identified by studying pre-steady-state and steady-state kinetics of the wild-type GA/maltose and Trp120 -->Phe GA/maltotriose reactions in H2O and D2O. Pre-steady-state fluorescence signal analysis was carried out to ascertain the relative intrinsic fluorescence of the enzyme intermediates. A three-step minimal pathway for oligosaccharide hydrolysis represented by E + Gx (k1) reversible (k-1) EGX (k2)reversible(k-2) EP (kcat)--> E + P is proposed. The first step, represented by the association constant K1 (k1/k-1), depicts the fast formation of enzyme-substrate complex and is the primary factor in fluorescence quenching. A 2.7-fold increase in K1 with D2O as solvent is observed with both enzymes due to the cumulative effect of deuterium on complex hydrogen bonding at the active site. The second step further quenches the enzyme fluorescence and is identified as the hydrolytic step, forming an enzyme-product complex. Both k2 and k-2 values show similar 2-fold decreases in D2O for both enzymes, consistent with the microscopic reversibility of the hydrolytic reaction. The solvent isotopic effect on the hydrolytic step is likely due to either abstraction of an exchangeable proton from the general acid Glu179 or directed addition of water to the oxocarbonium ion intermediate by the general base Glu400. No significant isotope effect was observed on the steady-state kcat value for wild-type GA with maltose, indicating a ronhydrolytic step as rate-limiting. The third step, a posthydrolytic rate-determining step, is the product release as evident from steady-state kinetics with wild-type and Trp120-->Phe GAs using alpha-D-glucosyl fluoride.     

Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori

Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylaseappeared by differential labeling to be in the active site.To test their functions, they were replaced by mutagenesis withAsn, Gln and Gln respectively, and kinetic parameters and pHdependencies of all enzyme forms were determined. Glu179 –Gln glucoamylase was not active on maltose or isomaltose, whilethe k_cat for maltoheptaose hydrolysis decreased almost 2000-foldand the K_M was essentially unchanged from wild-type glucoamylase.The Glu180 – Gln mutation drastically increased the K_Mand moderately decreased the k_cat with maltose and maltoheptaose,but affected isomaltose hydrolysis less. Differences in substrateactivation energies between Glu180 – Gln and wild-typeglucoamylases indicate that Glu180 binds D-glucosyl residuesin subsite 2. The Asp176 – Asn substitution gave moderateincreases and decreases in K_M and k_cat respectively, and thereforesimilar increases in activation energies for the three substrates.This and the differences in subsite binding energies betweenAsp176 – Asn and wild-type glucoamylases suggest thatAsp176 is near subsite 1, where it stabilizes the transitionstate and interacts with Trp120 at subsite 4. Glu179 and Asp176are thus proposed as the general catalytic acid and base ofpK_a 5.9 and 2.7 respectively. The charged Glu180 contributesto the high pK_a value of Glul79. Received May 25, 1989; accepted October 19, 1989.     

Artificial antibodies for affinity chromatography of homologous proteins: application to blood clotting proteins

OBJECTIVE: To determine whether pituitary down-regulation after gonadotropin-releasing hormone analogue (GnRH-a) administration can be accurately predicted by transvaginal ultrasonographic measurement of endometrial thickness. DESIGN: Prospective study. SETTING: An IVF unit of an academic medical center. PATIENT(S): One hundred eighty-one patients undergoing 265 IVF-ET treatment cycles using GnRH-a in the long protocol. MAIN OUTCOME MEASURE(S): Serum concentrations of E2 were determined, and endometrial thickness was measured by transvaginal sonography. The accuracy of endometrial thickness for predicting pituitary down-regulation was calculated. RESULT(S): Pituitary down-regulation, defined as a serum E2 concentration of < or = 55 pg/mL, was achieved in 77% (204 of 265) of the cycles. An endometrial thickness of < or = 6 mm was found in 92.2% (188 of 204) of cycles in which down-regulation was achieved. An estradiol level of < or = 55 pg/mL was present in 95.9% (188 of 196) of cycles with endometrial thickness of < or = 6 mm. CONCLUSION(S): A state of relative hypoestrogenism after GnRH-a administration, indicative of pituitary down-regulation, can be predicted with a high degree of accuracy by ultrasonographic measurement of endometrial thickness. Thus, routine testing for serum E2 concentration may be safely omitted. This may allow further simplification of IVF protocols and increase both cost-effectiveness and patients' convenience.