Thesis (Ph.D.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (Ph. D.)--Washington State University, May 2007. / Includes bibliographical references.

The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membraneous network that, through the cyclical release and re-uptake of Ca²⁺ into and from, respectively, the cytoplasmic space, regulates myofilament shortening and, therefore, muscle contraction. SR derived from the terminal cisternae (HSR) demonstrates the property of Ca²⁺-induced Ca²⁺ release. Upon attainment of a threshold intralumenal Ca²⁺ load, application of a small pulse of extralumenal Ca²⁺ stimulates the release of a pool of intralumenal Ca²⁺ via the ligand gated Ca²⁺ permeable pore of the Ca²⁺ release channel/ryanodine receptor complex. It was hypothesised that intralumenal Ca²⁺ regulates the opening of the release channel. HSR vesicles were purified from skeletal and cardiac muscle by a novel technique. Structural characterisation of these membranes demonstrated an enrichment of harvested fractions in the Ca²⁺ release channel and the intralumenal Ca²⁺ binding protein, calsequestrin. In radiometric studies, skeletal HSR vesicles were shown to bind ryanodine with high capacity at both low and high affinity sites, with 2 fold stimulation of Ca²⁺ accumulation by the polyorganic cation Ca²⁺ channel blocker, ruthenium red. HSR vesicles passively loaded Ca²⁺. Passive loading of HSR vesicles with Ca²⁺ was found to be non-linearly dependent upon the concentration of Ca²⁺ within the loading medium. This suggested the presence of 2 intralumenal Ca²⁺ binding sites with different affinities for Ca²⁺. A spectroscopic dual-wavelength assay of Ca²⁺ release was developed that took advantage of peculiar spectral properties of the metallochromic sensitive dye Antipyrylazo III. In the presence of mM MgATP and mM Mg2+ the initial fast phase of HSR Ca²⁺ was well resolved. Evidence was presented that initial rapid uptake was associated with high affinity binding to an intralumenal compartment. Ca²⁺ -induced Caz+ release was shown to occur with a threshold loading of intralumenal Ca²⁺. The intralumenal Ca²⁺ threshold for Ca²⁺-induced Ca²⁺ release was decreased in the presence of ryanodine. Ryanodine induced Ca²⁺ release was also dependent upon the amount of intralumenal Ca²⁺. Ryanodine was also shown to inhibit sustained Ca²⁺-induced Ca²⁺ release by apparent inhibition of the binding of Ca²⁺ to intralumenal sites. These results suggested that junctional state transitions of the Ca²⁺ channel and calsequestrin were interdependent. Purified mM and mM Ca²⁺ activated neutral protease isoforms selectively cleaved the Ca²⁺ channel into 410 and 150kDa peptides with limited proteolysis. This was demonstrated in both HSR vesicles and the purified Ca²⁺ release channel. A novel 88kDa protein was also shown to be fragmented by both CANP isoforms. The identity of this prominent HSR associated protein remains obscure. CANP fragmentation of HSR protein elevated passive and active 4^Ca²⁺ loading in vesicles. This indicated that selective structural modification of the cytoplasmic portion of the release channel modified the comformational states of a intralumenal Ca²⁺ binding compartment in HSR vesicles. In spectroscopic studies, CANP proteolysis of HSR proteins increased the sensitivity to Ca²⁺ and ryanodine-induced Ca²⁺ release through decreases in the required intralumenal Ca²⁺ threshold for release. These functional alterations coincided with apparent single site cleavage of the release channel. Further proteolysis of the initial 410 and 150kDa peptides was without further significant effect upon function. Based upon the hypothesis that primary sequences rich in proline (P), glutamate (E), aspartate (D), serine (S) and threonine (T) (PEST regions) are recognition sites for CANP binding to substrates, a search for PEST regions within the Ca²⁺ channel was undertaken. It was tentatively proposed that two PEST regions near the N-terminal of the Caz release channel may represent sites close to the CANP cleavage site. The results of this work were discussed in relation to a possible role of Ca²⁺-induced Ca²⁺ release in regulating the patterning of Ca²⁺ cytosolic transients. The frequency and amplitude of cytosolic Ca²⁺ transients appear to be important in regulating protein expression. The requirement of intralumenal Ca²⁺-induced Ca²⁺ release may be a means by which the cyclical uptake and release of Ca²⁺ during muscle relaxation and contraction can be coordinated. This coordination may define the patterning of cytosolic Ca²⁺ transients. The increased sensitivity to Ca²⁺-induced Ca²⁺ release by HSR after CANP treatment may represent a means by which the patterning of cytosolic Ca²⁺ transients can be altered to effect changes in protein synthesis. / Graduate and Postdoctoral Studies / Graduate

La tumeur maligne primitive du foie la plus fréquente est le carcinome hépatocellulaire. Le développement des cellules tumorales hépatiques dépend d'un influx calcique dont la base moléculaire, dans le foie, est constituée de TRPC6, des STIM1 et 2, et de Orai1. Nous avons recherché si le développement de ces tumeurs était accompagné d'un changement du profil d'expression de ces protéines. Nous avons montré que le canal TRPC6 est absent des hépatocytes humains sains mais fortement exprimé dans les tumeurs hépatiques, les foyers d'expression de TRPC6 étant visibles uniquement dans les zones tumorales du foie. Le niveau d'expression de TRPC6 dépend de certains facteurs de croissance et cette expression est directement corrélée à l'amplitude de l'entrée de calcium et à la prolifération cellulaire des hépatomes humains. STIM1 et Orai1 sont aussi impliqués dans cette relation, l'inhibition de l'expression de TRPC6, STIM1 ou Orai1 entraînant une baisse de l'expression des cyclines D1 dans ces cellules. Inversement, le blocage du cycle cellulaire va entraîner, de manière réversible (<4heures), une importante réduction de l'entrée de calcium. Les cultures primaires d'hépatocytes humains nous ont permis de confirmer ces résultats, démontrant ainsi la relation étroite entre entrée calcique et prolifération cellulaire. Nos derniers résultats ont montré que la base moléculaire de cette entrée dépend du type cellulaire utilisé mais que Orai1 en est la pièce centrale. Ce travail de thèse a permis d'élargir les connaissances sur la composition du complexe moléculaire formant l'entrée calcique directement impliquée dans la prolifération cellulaire et le développement de tumeurs hépatiques. / Hepatocellular carcinoma is the most frequent primary liver tumor. Tumoral cell proliferation depends on calcium influx which molecular basis in human liver is formed of TRPC6, STIM1 and 2, and Orai1. We have investigated whether tumors' development in human livers was accompanied by a change in the pattern of expression of these proteins. We have shown that TRPC6 channel is absent in healthy human hepatocytes but strongly expressed in human liver tumors, and TRPC6 expression is only detectable in the tumor zones. TRPC6 expression level depends on growth factors, and this expression is directly correlated with the amplitude of the calcium entry and cell proliferation of human hepatoma cell line. STIM1 and Orai1 also play a major role in this process and inhibition of the expression of TRPC6, STIM1 or Orai1 causes a decrease in cyclin D1 expression in human hepatoma cell line. Conversely, cell cycle block results in a large decrease in calcium influx that quickly reversed in less than 4 hours after cell cycle release. Primary cultures of human hepatocytes allowed us to confirm these results, emphasizing the tight relation between calcium influx and cell proliferation. Our recent data strongly suggested that the molecular basis of this calcium entry varies from one cell type to another, with Orai1 as the core of this plasma membrane complex. In conclusion, this thesis has expanded the knowledge about the actors of calcium entry and their role in the proliferation of both liver cells and liver cancer development

The production of reactive oxygen species (ROS) may be a contributor to the progression of sarcopenia. Sarcopenia is a generic term for the loss of skeletal muscle mass, quality and strength. ROS are usually produced by radiation, but are also the byproducts of aerobic metabolism. ROS have been found to mediate various pathological conditions in a variety of tissues, to cause oxidative damage to DNA, proteins, and lipids with advancing age, and is presumably a major factor contributing to changes associated with aging. The purpose of this investigation was to determine whether the sarcoplasmic reticulum (SR) of muscle from aged animals are more susceptible to the deleterious effects of ROS. Using isolated gastrocnemius SR vesicles extracted from adult (12m) and aged (27m) male Brown Norway-Fischer 344 hybrid rats, Ca2+ uptake and release measurements were obtained. The data showed that there was a 33% difference between aged and adult gastrocnemius mass. When gastrocnemius mass was corrected for body mass, the differences was ~20% between the two groups. A 20% decrease in SR Ca2+ uptake rate was noted in aged animals. HOCl also, decreased uptake by similar extents in both groups. This result suggest that the Ca2+ pump's response to ROS are similar in both groups. AgNO3 -induced and H2O2 -induced release in aged animals was 17.94 and 7.39 nmol/mg/min and in adult animals was 30.46 and 7.18 nmol/mg/min, respectively. H2O2-induced release, when expressed as a percent of AgNO3-induced release was increased in aged animals by 54%. The results suggest that the release channel of aged muscle appears to be more sensitive to ROS. In conclusion, the data support the theory that aged animal skeletal muscle is more susceptible to the adverse effects of ROS. / Master of Science

Isotopic and spectrophotometric assays show that micromolar concentrations of heavy metal ions (particularly Ag⁺ and Hg²⁺) stimulate ATPase activity but inhibit Ca²⁺ uptake in isolated SR vesicles. Both effects are caused by increased Ca²⁺ permeability of the membrane, apparently the result of activation of the Ca²⁺ release system of the vesicles due to heavy metal binding to a critical sulfhydryl group associated with the Ca²⁺ release channels. CU²⁺catalyzed co-oxidation of this sulfhydryl with exogenous cysteine to form a mixed disulfide also results in activation of the Ca²⁺ release system. The rate and extent of Cu²⁺/cysteine-induced release is maximal at physiological pH and is inhibited by local anaesthetics and Mg²⁺, suggesting that the redox state of this sulfhydryl may play a role in activation of the Ca²⁺ release system of intact muscle. Modification of the SR with the proteolytic enzyme trypsin also increases the Ca²⁺ permeability of the SR, and enhances the rate of Ca²⁺ release activated by cAMP, doxorubicin, Hg²⁺, and Cu²⁺/cysteine. The rates of release activated by all reagents are stimulated by a factor of five after five minutes exposure to trypsin. Hg²⁺- and Cu²⁺/cysteine-activated release are not stimulated further, while cAMP- and doxorubicin-activated release continue to increase up to maximum of 20-fold stimulation after 15 minutes exposure to trypsin. Inhibitors of the Ca²⁺ release system such as Mg²⁺ and ruthenium red still inhibit release from proteolytically modified SR, and the binding affinities of activators and inhibitors to their sites are not significantly altered by proteolysis; only the rates of Ca²⁺ transport are affected. The most probable mechanisms of tryptic stimulation of Ca²⁺ release are (1) removal of a regulatory protein or subunit of the Ca²⁺ release system, making more channels available for transport; (2) increasing the single channel unitary conductance; (3) increasing the open time of activated channels. The biphasic character of proteolytic stimulation of cAMP- and doxorubicin-activated release (as opposed to monophasic stimulation of Hg²⁺- and Cu²⁺/cysteine-activated release} suggests that more than one of the above parameters are involved in tryptic stimulation of the Ca²⁺ release system.

<p>The partitioning of translation to the outer membrane of the endoplasmic reticulum is a problem that has been the subject of inquiry since the discovery of the ribosome. The large degree to which ribosomes were found to be tethered to the membrane led to intense investigation of a series of related questions regarding the identity of those mRNAs that are translated on the endoplasmic reticulum, and the functions of that localization in cell stress. In this dissertation, I approach each of these questions in turn and work to reconcile my observations with those models that have been previously proposed. A theme of this work is the application of modern methods, particularly deep sequencing technology, to address problems that had largely been considered solved. The most prominently featured method is ribosome profiling, which is paired with classical biochemical and cell biological techniques. I arrive at several conclusions: 1) a significant fraction of all mRNAs is well represented on the endoplasmic reticulum membrane, 2) the properties of translation diverge substantially between membrane-associated and free ribosomes, and 3) the compartmentalization of translation can serve as an important variable in cell stress.</p> / Dissertation

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX), infamously known as the “red devil,” is considered the most effective antineoplastic drug utilized in oncologic practice today. However, its clinical use is hampered due to cumulative, dose-dependent cardiotoxicity, which can lead to reduced quality of life, irreversible heart failure and death. The mechanisms involved in the pathogenesis of cardiotoxicity have not been fully elucidated, but have previously been demonstrated to involve oxidative stress, calcium dysregulation and mitochondrial dysfunction. Since the mitochondria play a critical role in generation of reactive oxygen species, the maintenance of calcium homeostasis and are the most extensively damaged by DOX, they have become the main focus of novel therapeutic interventions. The morphology and function of these dynamic organelles are regulated in part by mitochondrial fission and fusion events, as well as mitochondrial quality control systems. Since mitochondrial morphology is often associated with crucial cellular functions, this study aimed to investigate the long-term effects of DOX on mitochondrial dynamics and the mitochondrial quality control systems, mitophagy and the ubiquitin-proteasome pathway (UPP). Additionally, since the mitochondria and the endoplasmic reticulum (ER) are two interconnected organelles, and both play a role in maintaining calcium homeostasis, this study further assessed the effects of chronic DOX treatment on ER function and calcium status. Materials and Methods: In order to fully establish the effect of chronic DOX treatment in vitro, two cardiac cell lines were utilized in this study. H9C2 cardiomyoblasts and humanderived Girardi heart cells were cultured under standard culture conditions until ± 70-80% confluency was reached, where after treatment commenced. Cells were treated daily with 0.2 and 1.0 μM of DOX for 96 and 120 hours in order to simulate chronic, cumulative cardiotoxicity. Cell viability and apoptotic cell death were assessed with the MTT assay and Caspase Glo 3/7 assays, respectively. The expression of proteins involved in mitochondrial dynamics, mitochondrial biogenesis, the ubiquitin-proteasome pathway, mitophagy and ER stress were determined with Western blotting. Organelle morphology was visualized with fluorescence microscopy, and flow cytometry was used to assess mitochondrial and ER load. In order to determine the oxidative capacity, stress and status within the cells following treatment, the Oxygen radical absorbance capacity (ORAC), Thiobarbituric acid reactive substances (TBARS) and Glutathione (GSH) assays were employed respectively. Finally, intracellular and mitochondrial calcium was assessed and quantified with superresolution structured illumination microscopy (SR-SIM) and flow cytometry respectively. Results: DOX significantly reduced cell viability and increased apoptosis in both in vitro cardiac cell models. This study further demonstrated that the expression of mitochondrial fusion proteins, Mfn 1 and Mfn 2 were significantly downregulated, whilst the regulators of fission, Drp1 and hFis1, were significantly elevated, therefore shifting the balance of mitochondrial dynamics towards fission. Unopposed and elevated mitochondrial fission was clearly evident from the morphology of these organelles, which displayed short, highly fragmented mitochondria with a dispersed network following treatment. Chronic DOX also downregulated the regulator of mitochondrial biogenesis, PGC-1α, thus inhibiting the formation of new, functional mitochondria. The E3 ligases, MARCH5 and Parkin were highly upregulated following treatment, indicating activation of the UPP and mitophagy. Although chronic DOX stimulated K48 ubiquitination following treatment, it inhibited the catalytic activity of the 26S proteasome, therefore blocking proteasomal degradation. Although the antioxidant capacity (measured as ORAC) was significantly enhanced by both concentrations of DOX, an increase in oxidative stress status was shown following DOX treatment. In this regard lipid peroxidation significantly increased, while redox status of the endogenous antioxidant, glutathione, significantly decreased. Additionally chronic DOX treatment induced ER stress, which lead to an increase in cytosolic and mitochondrial calcium. In response to ER stress, the unfolded protein response (UPR) was then stimulated. Discussion: Results from this study indicate that chronic DOX treatment disrupts the balance of mitochondrial dynamics, favouring mitochondrial fission. Mitochondrial fragmentation is mediated by the downregulation of fusion proteins regulated by the E3 ubiquitin ligase, MARCH5 as well as by the increase in mitochondrial calcium. Mitochondrial fission results in mitophagy, an adaptive response to protect the cardiac cell against damaged mitochondria. This study also indicates that during chronic DOX-induced cardiotoxicity ER stress and the UPR are induced, which is possibly responsible for the disruption in calcium homeostasis. The inhibition of mitochondrial biogenesis coupled with elevated mitophagy as observed in this chronic study, elucidates a plausible mechanism whereby DOX induces mitochondrial dysfunction. Unregulated mitochondrial fragmentation and inhibited mitochondrial biogenesis are known to regulate various cardiomyopathies, therefore since both these effects are induced by chronic DOX treatment suggests a mechanism whereby cardiotoxicity, and ultimately heart failure are produced. This study provides new insight into the role of chronic DOX plays in altering mitochondrial dynamics and mitochondrial quality control systems. Further investigations targeted at limiting mitochondrial fission may reduce the cardiovascular side effects associated with DOX. / AFRIKAANSE OPSOMMING: Inleiding: Doksorubisien (DOX), ook bekend as die “rooiduiwel,” word beskou as die mees effektiewe anti-neoplastiese middel wat tans in onkologie praktyke gebruik word. Die kliniese gebruik hiervan word gerem deur die kumulatiewe dosis-afhanklike kardiotoksisiteit wat tot verlaagde lewenskwaliteit, onomkeerbare hartversaking, en tot die dood kan lei. Die meganismes wat by die kardiotoksiese patogenese betrokke is, is nog onbekend, maar die meganisme het moontlik te doen met oksidatiewe stres, kalsiumwanregulering en mitochondriale wanfunksionering. Omrede die mitochondria ‘n kritieke rol in die vorming van reaktiewe suurstofspesies speel, asook die handhawing van kalsiumhomeostase en die mees beskadigde organelle deur DOX, het die hooffokus na nuwe terapeutiese intervensies verskuif. Die morfologie en funskie van hierdie dinamiese organelle word gereguleer deels deur mitochondriale fragmentering en fussie, asook mitochondriale kwaliteitsbeheersisteme. Omrede mitochondriale morfologie geassosieer is met noodsaaklike sellulêre funksies, het hierdie studie gepoog om die langtermyneffkte van DOX op mitochondriale dinamika en die mitochondriale kwaliteitsbeheersisteme, mitofagie en die ubikwitien-proteosoomweg (UPW) te ondersoek. Siende dat die mitochondria en die endoplasmiese retikulum (ER) twee interverweefde organelle is, en beide ‘n rol speel in die handhawing van kalsiumhomeostase, het hierdie studie verder die effekte van chroniese DOX behandeling op ER funksie en kalsiumstatus ondersoek. Materiaal en Metodes: Om die effek van chroniese DOX behandeling in vitro te verstaan in hierdie studie, is twee hartsellyne gebruik. H9C2 kardiomioblaste en menslike Girardi hartselle is onder standaardtoestande tot ± 70-80% konfluensie bereik is gekweek, waarna behandeling begin is. Selle is daagliks met 0.2 en 1.0 μM DOX vir 96 en 120 uur behandel om chroniese en kumulatiewe kardiotoksisiteit n ate boots. Selvatbaarheid en apoptotiese seldood is onderskeidelik ondersoek deur middel van die MTT en Caspase Glo 3/7 toetse. Die proteïenuitdrukking betrokke by mitochondriale dinamika, mitochondriale biogenese, die ubikwitien-proteosoom weg, mitofagie en ER stres is deur middel van westerse afblatting bepaal. Organelmorfologie is deur middel van fluoresensie mikroskopie gevisualiseer, en vloeisitometrie was gebruik om die aantal mitochondria en ER lading te bepaal. Om die oksidatiewe kapasiteit, stres en status binne die selle na behandeling te bepaal, is die ORAC, TBARS en GSH toetse onderskeidelik gebruik. Laastens was die intrasellulêre en mitochondriale kalsium ondersoek en gekwantifiseer met superresolussie gestruktureerde illuminasie mikroskopie (SR-SIM) en vloeisitomerie. Resultate: DOX het selvatbaarheid betekenisvol verlaag en apoptose in beide in vitro kardiale selmodelle verhoog. Hierdie studie het verder aangetoon dat die uitdrukking van mitochondriale fussie proteïene, Mfn 1 en Mfn 2 betekenisvol afgereguleer is, terwyl die reguleerders van fragmentering, Drp1 en hFis1, betekenisvol verhoog is en daardeur die balans van mitochondriale dinamika na fussie verskuif. Onverhinderde en verhoogde mitochondriale fragmentering is duidelik sigbaar deur die morfologie van die organelle, wat as kort, hoogsgefragmenteerde mitochondria met ‘n verspreide netwerk na behandeling vertoon. Chroniese DOX het ook die mitochondriale biogenese reguleerder, PGC-1α, afgereguleer en daardeur die vorming van nuwe, funksionele mitochondria geinhibeer. Die E3 ligase, MARCH5 en Parkin is hoogs opgereguleer na behandeling, wat aktivering van UPW en mitofagie aantoon. Alhoewel chroniese DOX K48 ubikwitinering na behandeling gestimuleer het, het dit die katalitiese aktiwiteit van die 26S proteasoom geinhibeer en dus die proteosomale degradasie geblokkeer. Antioksidantkapasiteit en oksidatiewe status was betekenisvol na behandeling wat gevolglik tot hoë vlakke oksidatiewe skade binne die selle gelei het. Addisioneel het chroniese DOX behandeling ER stres geïnduseer wat tot ‘n toename in sitosoliese en mitochondriale kalsium gelei het. In reaksie op die ER stres is die UPW gestimuleer. Bespreking: Resultate van hierdie studie het aangetoon dat chroniese DOX behandeling die balans van mitochondriale dinamika onderbreek en sodoende mitochondriale fragmentering bevoordeel. Mitochondriale fragmentering word gemediëer deur die afregulering van fussie proteïene wat deur die E3 ubikwitienligase, MARCH5, gereguleer word, en ook deur die toename in mitochondriale kalsium. Mitochondriale fragmentering induseer mitofagie, ‘n aanpassingsreaksie om die hartselle teen beskadigde mitochondria te beskerm. Hierdie studie toon verder ook dat gedurende chroniese DOX-geïnduseerde ER stres, word die UPW ook geïnduseer, wat moontlik dan verantwoordelik is vir die ontwrigting van kalsiumhomeostase. Die inhibering van mitochondriale biogenese gekoppel met verhoogde mitofagie soos waargeneem in hierdie studie, verklaar ‘n moontlike meganisme waardeur DOX mitochondriale wanfunksionering veroorsaak. Ongereguleerde mitochondriale fragmentering en geinhibeerde mitochondriale biogenese is bekend om verskeie kardiomiopatieë te reguleer. Omrede beide hierdie effekte geinduseer word deur chroniese DOX behandeling kan dit moontlik ‘n meganisme voorstel waarby kardiotokiese en uiteindelik hartversaking ontwikkel. Hierdie studie bied nuwe insig in die rol wat chroniese DOX speel in die wysiging van mitochondriale- dinamika en kwaliteitskontrole sisteme. Verdere ondersoeke wat die mitochondriale fragmentering kan verminder mag moontlik die kardiovaskulêre newe-effekte wat met DOX behandeling geassosieer is, verlaag. / National Research Foundation (NRF)

In mammalian cells newly synthesised proteins are translocated across the ER membrane and their subsequent folding is facilitated by an array of folding factors present in the lumen. These include the lectins calreticulin and calnexin, which form complexes with ERp57 to generate glycoprotein specific molecular chaperones. ERp57 is a member of the protein disulphide isomerase (PDI) family and its binding to ER lectins can be reconstituted in vitro. I have exploited this approach to define the regions of ERp57 that are necessary and sufficient for its specific interaction with calreticulin and calnexin. Truncated forms of ERp57, chimeric proteins containing various domains of ERp57 and PDI (which does not interact with calreticulin) and ERp57 b' domain point mutants have been constructed. By analysing the interactions of ERp57 derivatives with calreticulin using both cross-linking and binding assays I have been able to provide detailed insights into the molecular basis for the specific assembly of these components within the ER lumen. My results indicate that the b and b' domains of ERp57 are necessary, but not sufficient for binding to both calreticulin and calnexin. The more stringent binding assay revealed that the a' domain of ERp57 significantly enhanced binding to biotin-tagged calreticulin. The ERp57 C-terminal extension also increased binding to biotin-tagged calreticulin, perhaps by playing a role in the overall stability of the ERp57. In addition, the ERp57 b' domain point mutants show that certain amino acids in this domain, in particular residues F280, V283 and F299, may be crucial for binding to calreticulin, consistent with the principal lectin-binding site being located in the b' domain. However, the binding region clearly extends into other domains, in particular the b and a' domains.

No description available.