This study reports the isolation, purification and partial characterisation of the ostrich serpin, a2AP, as well as its target enzyme, ostrich plasmin, in its active and inactive proenzyme, viz. plasminogen, forms. Three different procedures were undertaken to isolate and purify ostrich a2AP. The first one involved L-lysine-Sepharose chromatography, ammonium sulfate fractionation, ion-exchange chromatography on Toyopearl Super-Q 650S, and ostrich plasminogen-Sepharose affinity chromatography. The second procedure replaced the latter chromatographic step with gel filtration on Sephadex G-200 and hydroxylapatite chromatography, while the third one employed instead the theoretically more efficient LBSI-Sepharose chromatographic step. The third procedure yielded purified ostrich a2AP, but the degree of purity and yield were relatively low. Ostrich plasminogen was highly purified after L-lysine-Sepharose chromatography and ostrich plasmin was obtained by the urokinase-activation of the purified ostrich plasminogen Ostrich a2AP revealed an Mr of 77-84 K and two isoelectric forms of pI 3.85 and 6.18. Nterminal sequence analysis showed ostrich a2AP to have only 2 out of 11 residues in common with both those of human and bovine a2AP. Ostrich a2AP showed the largest inhibitory effects on ostrich plasmin, followed by comm. bovine chymotrypsin, trypsin and plasmin, in that order, and it appeared to be a much less potent plasmin inhibitor than bovine aprotinin, but a much more potent one than the synthetic inhibitors, DFP and EACA. Ostrich plasminogen showed an Mr of 92 K and multiple isoelectric forms (~7) in the pI range 6.01-9.18, with a major one of pI 6.01. It showed a total of 775 amino acid residues and its N-terminal sequence showed ~53 percent identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin revealed an Mr of 78 K, two isoelectric forms of pI 4.07 and 6.01, and a total of 638 amino acid residues. N-terminal sequence analysis showed that 2-4 residues are identical to the 5 of human, cat, dog, rabbit, and ox plasmins. The pH and temperature optima of ostrich plasmin were determined as 8.0 and 40 oC, respectively. The thermodynamic and kinetic parameters of ostrich plasmin were computed, and plasmin was shown to prefer Lys to Arg residues in the S1 position. In conclusion, ostrich a2AP, plasminogen and plasmin showed definite similarities to their mammalian counterparts, but there were also significant differences.

Doctor of Philosophy / Biochemistry / Michael R. Kanost / Insect innate immune responses include prophenoloxidase activation and antimicrobial peptide production. These responses involve extracellular serine proteinase cascades that are regulated by serpins. This work involved the study of serine proteinase 16 (HP16) and serpin-3 from hemolymph of the tobacco hornworm, Manduca sexta. HP16 has an amino-terminal domain with no similarity to any characterized protein and a carboxyl-terminal S1 family serine proteinase domain. HP16 levels in plasma were highest during the wandering, prepupal, and pupal stages. HP16 mRNA levels in fat body were highest at the wandering stage. Injection of bacteria into fifth instar larvae stimulated HP16 expression. To further characterize and investigate the biological function of HP16, recombinant proteins for proHP16, two HP16 mutants, the amino-terminal domain (NT16), and three NT16 mutants were purified. Recombinant HP16 was cleaved at the predicted activation site during expression, and its amino-terminal and catalytic domains remained connected by a disulfide bond. ProHP16 in plasma was apparently activated in the presence of the microbial elicitor, zymosan. Recombinant HP16 formed a complex with serpin-1Z, indicating that it was catalytically active, but no other natural or artificial substrates were identified. Analysis of NT16 and NT16 mutants led to the discovery that multiple disulfide bond arrangements were formed in the recombinant amino-terminal domain of HP16. This work furthered the understanding of HP16 and laid a foundation for subsequent experiments involving the proteolytic activity, regulation, and biological function of HP16. Active serine proteinases in insect hemolymph are often regulated by serpins. Immunoaffinity chromatography was used to identify plasma proteinases that are inhibited by serpin-3. Four serpin-3-proteinase complexes purified from plasma were identified by immunoblot analysis as serpin-3 complexes with HP8, PAP-1, PAP-2, and PAP-3. MALDI-TOF/TOF or ESI-MS/MS analysis after separation by 1D- or 2D-PAGE confirmed serpin-3 complex formation with HP8, PAP-1, and PAP-3. ProHP8 in plasma was activated by exposure to the β-1,3-glucan curdlan and inhibited by serpin-3. Purified recombinant serpin-3 and active HP8-Xa formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.

No description available.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Analytical separations form the bulk of experiments in both research and industry. The choice of separation technique is governed by the characteristics of the analyte and purpose of separation. Miniaturization of chromatographic techniques enables the separation and purification of small volume samples that are often in limited supply. Capillary electrophoresis and immunoaffinity chromatography are examples of techniques that can be easily miniaturized with minimum loss in separation efficiency. These techniques were used in the experiments presented in this dissertation. Chapter 1 discusses the underlying principles of capillary electrophoresis and immunoaffinity chromatography. In the second chapter, the results from immunoaffinity chromatography experiments that utilized antibody-coated magnetic beads to purify serine proteases and serine protease inhibitors (serpins) from A. gambiae hemolymph are presented and discussed. Serine proteases and serpins play a key role in the insect innate immunity system. Serpins regulate the activity of serine proteases by forming irreversible complexes with the proteases. To identify the proteases that couple to these serpins, protein A magnetic beads were coated with SRPN2 antibody and then incubated with A. gambiae hemolymph. The antibody isolated both the free SRPN2 and the SRPN2-protease complex. The purified proteases were identified by ESI-MS from as few as 25 insects. In Chapter 3, an integrated glass/PDMS hybrid microfluidic device was utilized for the transportation and lysis of cells at a high throughput. Jurkat cells were labeled with 6-CFDA (an internal standard) and DAF-FM (a NO specific fluorophore). Laser-induced fluorescence (LIF) detection was utilized to detect nitric oxide (NO) from single Jurkat cells. The resulting electropherograms were used to study the variation in NO production following stimulation with lipopolysaccharide (LPS). 3 h LPS-stimulation resulted in a two fold increase in NO production in both bulk and single cell analysis. A comparison of bulk and single cell NO measurements were performed and the average NO production in single cells compared well to the increase measured at the bulk cell level. Chapter 4 discusses the preliminary experiments with a T-shaped microfluidic device that exploit the property of poly(dimethylsiloxane) (PDMS) as an electroactive polymer (EAP), to enhance fluid mixing. EAPs deform when placed in an electric field. A thin layer of PDMS was sandwiched between chrome electrodes, positioned on the horizontal arms of the T design, and the electrolyte-filled fluidic channel. A potential difference across the PDMS layer caused it to shrink and stretch, thereby increasing the channel volume. The electrodes were actuated at 180[degrees] out of phase and this caused the fluid stream in the vertical channel to fold and stretch resulting in enhanced contact surface area and shorter diffusion distances of the fluid, thereby improving mixing efficiency. All the experiments presented in this dissertation demonstrate the application of miniaturized chromatographic techniques for the efficient analysis of small volume biological samples.

Abstract not available

Abstract not available

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Orientador: Prof. Dr. Luciano Puzer / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / As calicreinas teciduais humanas (KLKs) compreendem uma familia de quinze serino proteases encontradas em uma diversidade de fluidos e tecidos biologicos. Estas enzimas sao identificadas como possuindo papel em diferentes doencas como Alzheimer, cancer, dermatite atopica, esclerose multipla, Parkinson, psoriase e outras. Existe, portanto, uma crescente demanda por inibidores especificos para cada uma das calicreinas e este e o objetivo do nosso grupo de pesquisa na UFABC. Neste trabalho pretendemos gerar inibidores para as calicreinas teciduais humanas 3, 5 e 7, utilizando bibliotecas baseadas em duas serpinas diferentes: uma expressando a forma Pittsburgh do inibidor de proteinase-¿¿1 (IP-¿¿1 M358R), randomizada nos residuos 352-356 (P7-P3); e outra expressando a serpina bacteriana vioserpina, randomizada nos residuos 343-347 (P3-P2f). A abordagem do phage display foi eficaz para gerar as bibliotecas e o protocolo de bioselecao utilizado adequado para enriquecer diversas variantes reativas. Na selecao da biblioteca do IP-¿¿1 M358R, consensos PSEAL e PSRIL foram observados, para KLK5 e KLK7, respectivamente, e varias das sequencias selecionadas exibiram maiores taxas de inibicao para ambas as calicreinas, quando comparadas a molecula molde (IP-¿¿1 M358R). A variante HDVIL e o consenso PSRIL foram identificados como sendo altamente seletivos para a KLK7, com constantes de segunda ordem 14 e 33 vezes maiores que as para KLK5. Pudemos realizar uma selecao efetiva da biblioteca de vioserpina contra a KLK7, cujas variantes enriquecidos demonstraram uma preferencia geral pelo aminoacido Serina ocupando as posicoes P3, P1f, P2f e P1, seguido por uma Tirosina, tambem preferida em P2. A tecnica de phage display foi, portanto, eficiente como base para um estudo de especificidade, e para o desenvolvimento de melhores e mais especificos inibidores para as Calicreinas Teciduais Humanas, e pode ser utilizada para o desenvolvimento de novas bibliotecas, com outras regioes da RCL randomizadas, ou mesmo baseadas em outras serpinas. / The human tissue kallikreins (KLKs) comprise a family of fifteen serine proteases found in a diversity of biological fluids and tissues. These enzymes are identified as having a role in different diseases such as Alzheimer's, cancer, atopic dermatitis, multiple sclerosis, Parkinson's, psoriasis, and others. Thus there is a growing demand for specific inhibitors for each of these kallikreins, and this is the aim of our group at UFABC. In this work we intended to generate inhibitors for the human tissue kallikreins 3, 5 and 7, using libraries based on two different serpins: one expressing the Pittsburgh form of the human serpin á1-proteinase inhibitor (á1-PI M358R), randomized at residues 352-356 (P7-P3); and another one expressing the bacterial vioserpin, randomized at residues 343-347 (P3-P2¿). The phage display approach was effective to generate the libraries and the biopanning protocol used suitable to enrich numerous reactive variants. On the á1-PI M358R selection, loose consensus of PSEAL and PSRIL were observed, for KLK5 and KLK7, respectively, and several of the selected sequences exhibited higher inhibition rates when compared to the template molecule for both kallikreins. The variant HDVIL and consensus PSRIL were found to be highly selective for the KLK7, with second order constants 14- and 33-fold higher than the ones for KLK5. We could only perform an effective selection with the vioserpin library for the KLK7, whose enriched variants demonstrated a general preference for the amino acid Serine occupying the positions P3, P1¿, P2¿ and P1, followed by a Tyrosine, also preferred on the P2. The phage display approach was therefore effective as basis for a specificity study, and for the development of improved, more specific inhibitors for the Human Tissue Kallikreins, and can be used to develop new libraries, with other randomized RCL regions, or even based on other serpins.

<p>The serpin α-1 proteinase inhibitor (API) normally only impacts the coagulation cascade through its ability to inactivate factor XIa. However, the point mutation (Met to Arg) at position 358 results in a potent thrombin inhibitor, API M358R. This mutation also enhances this serpin’s ability to inhibit the anticoagulant protein, activated protein C (APC) and hence this property limits its therapeutic potential. As a result, various modifications to this protein have been engineered in order to enhance its specificity towards thrombin. Previously, the Heparin Cofactor II (HCII) N-terminal tail, HCII 1-75, which binds exosite 1 of thrombin, was tethered to the N-terminus of API M358R, creating HAPI M358R. Although this change did not alter anti-APC activity, it did augment the anti-thrombin activity of API M358R. In addition, further changes in the reactive center loop, the region that interacts with the thrombin active site, resulted in a significant reduction in APC activity while maintaining antithrombotic activity similar to HAPI M358R; this variant was termed HAPI RCL5.</p> <p>Preliminary experiments were performed with the C-terminal tridecapeptide of Hirudin Variant 3 (HV3) to determine its exosite 1 binding capacity compared to HCII 1-75. Three different variants of this peptide were tested: one with a hexahistidine tag (H₆HV3_54-66), another that also had a hexa-glycine C-terminal addition (H₆HV3_54-66G₆) and a third without either addition. All were found to bind exosite 1 with a greater affinity than HCII 1-75. Thus, the H₆HV3_54-66G₆peptide was fused to API M358R and API RCL5 in hopes of creating an inhibitor with heightened specificity compared to HAPI M358R and HAPI RCL5, respectively.</p> <p>HV3API M358R and HV3API RCL5 were expressed in a bacterial system and purified by nickel-chelate and ion exchange chromatography. Second order rate constants for the inhibition of thrombin and APC by the API variants and fusion proteins were determined. The K₂ values for α-thrombin inhibition ranged from 186 M^-1min^-1 to 22 M^-1min^-1 with an order of inhibitory potency observed as follows: HAPI M358R > HAPI RCL5 > HV3API M358R > HV3API RCL5>API RCL5 > API M358R.</p> <p>The ability of recombinant chimeric serpins to bind thrombin exosite 1 in a manner independent of RCL-thrombin active site interactions was also investigated through competitive inhibition of the binding of active site-inhibited thrombin to immobilized HCII 1-75. It was found that the order of exosite 1 binding affinity was HV3API RCL5 > H₆HV3_54-66G₆> HCII 1-75 > HAPI RCL5. Our results indicate that fusing the C-terminal tridecapeptide of HV3 to API variants enhanced their ability to inhibit thrombin, but to a lesser extent than fusing the N-terminal 75 residues of HCII. This finding likely reflects a requirement for the exosite 1-binding motif of the fusion protein to bind exosite 1 in a way that allows for subsequent optimal active site attack on the RCL by the serpin moiety of the fusion protein. In general, this work provides a second novel example of how the activity of a thrombin-inhibitory serpin can be enhanced by fusion to an exosite-1 binding motif.</p> / Master of Health Sciences (MSc)

Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA. We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3. The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.