Establishment and Characterization of Amitrole-Induced Mouse Thyroid Adenomatous Nodule-Derived Cell Lines.

Background: Thyroid cancer cell lines have been of great value for the study of thyroid cancer. However, the availability of benign thyroid adenoma cell lines is limited. Methods: Cell lines were established from thyroid adenomatous nodules that developed in mice treated with the goitrogen amitrole. Expression of epithelial, mesenchymal, and thyroid markers of these established cell lines was determined, and the effect of lentivirus-transduced overexpression of NKX2-1, a master regulator of thyroid development, on the thyroid marker expression was examined. Signal transduction and cell proliferation were evaluated after treatment with insulin-like growth factor-I (IGF-I) and the selective IGF-I receptor (IGF-IR) inhibitor NVP-ADW742. Xenograft studies were performed to examine tumorigenicity of the cells in mice. Whole-genome sequencing (WGS) was used to comprehensively determine the genetic mutations in the established two cell lines. Results: Five mouse thyroid adenomatous nodules-derived cell lines named CAT (cells from amitrole-treated thyroids) were established. Among these, two cell lines, CAT458/458s (CAT458s: a subline of CAT458) and CAT459, were found to be positive for epithelial markers and negative for a mesenchymal marker. NKX2-1-positive CAT459 cells showed higher messenger RNA (mRNA) expression of some thyroid differentiation markers than NKX2-1-negative CAT458s cells, and NKX2-1 overexpression increased and/or induced their expression. IGF-I signaling was transduced in thyrotropin receptor (Tshr)-negative CAT458s and 459 cells, and NVP-ADW742 suppressed their proliferation. No tumors developed in mice after subcutaneous injection of CAT458s or 459 cells. The WGS analysis revealed the presence of missense mutations in the tumor suppressor genes such as Polk (encoding DNA polymerase kappa) and Tgfb1 (encoding transforming growth factor beta 1), while no mutations were found in the prominent thyroid cancer-related genes Braf, Trp53 (encoding p53), and Tert (encoding telomerase reverse transcriptase). Conclusions: Two mouse thyroid adenomatous nodule-derived cell lines with different thyroid differentiation marker expression were established. NKX2-1 induced partial differentiation of these cell lines. They lacked tumorigenicity and prominent gene mutations involved in thyroid cancer development, while missense mutations were found in some tumor suppressors as revealed by WGS. The CAT458s and 459 provide a new tool to further clarify the process of thyroid multistep carcinogenesis and differentiation.

Anopheles salivary apyrase regulates blood meal hemostasis and drives malaria parasite transmission.

Mosquito salivary proteins play a crucial role in regulating hemostatic responses at the bite site during blood feeding. In this study, we investigate the function of Anopheles gambiae salivary apyrase (AgApyrase) in Plasmodium transmission. Our results demonstrate that salivary apyrase interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protein previously shown to be required for Plasmodium transmission. Microscopy imaging shows that mosquitoes ingest a substantial amount of apyrase during blood feeding which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. Supplementation of Plasmodium infected blood with apyrase significantly enhanced Plasmodium infection in the mosquito midgut. In contrast, AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammal host, underscoring the potential for new strategies to prevent malaria transmission.

The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel VHH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of VHH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

The in-silico evaluation of important GLUT9 residue for uric acid transport based on renal hypouricemia type 2.

Uric acid is the end product of purine metabolism. Uric acid transporters in the renal proximal tubule plays a key role in uric acid transport. Functional abnormalities in these transporters could lead to high or low levels of uric acid in the blood plasma, known as hyperuricemia and hypouricemia, respectively. GLUT9 has been reported as a key transporter for uric acid reuptake in renal proximal tubule. GLUT9 mutation is known as causal gene for renal hypouricemia due to defective uric acid uptake, with more severe cases resulting in urolithiasis and exercise induced acute kidney injury (EIAKI). However, the effect of mutation is not fully investigated and hard to predict the change of binding affinity. We comprehensively described the effect of GLUT9 mutation for uric acid transport using molecular dynamics and investigated the specific site for uric acid binding differences. R171C and R380W showed the significant disruption of the structure not affecting transport dynamics whereas L75R, G216R, N333S, and P412R showed the reduced affinity of the extracellular vestibular area towards urate. Interestingly, T125 M showed a significant increase in intracellular binding energy, associated with distorted geometries. We can use this classification to consider the effect mutations by comparing the transport profiles of mutants against those of chemical candidates for transport and providing new perspectives to urate lowering drug discovery using GLUT9.

Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

Identification of a dysfunctional exon-skipping splice variant in GLUT9/SLC2A9 causal for renal hypouricemia type 2.

Renal hypouricemia (RHUC) is a pathological condition characterized by extremely low serum urate and overexcretion of urate in the kidney; this inheritable disorder is classified into type 1 and type 2 based on causative genes encoding physiologically-important urate transporters, URAT1 and GLUT9, respectively; however, research on RHUC type 2 is still behind type 1. We herein describe a typical familial case of RHUC type 2 found in a Slovak family with severe hypouricemia and hyperuricosuria. Via clinico-genetic analyses including whole exome sequencing and in vitro functional assays, we identified an intronic GLUT9 variant, c.1419+1G>A, as the causal mutation that could lead the expression of p.Gly431GlufsTer28, a functionally-null variant resulting from exon 11 skipping. The causal relationship was also confirmed in another unrelated Macedonian family with mild hypouricemia. Accordingly, non-coding regions should be also kept in mind during genetic diagnosis for hypouricemia. Our findings provide a better pathogenic understanding of RHUC and pathophysiological importance of GLUT9.

Camel nanobodies broadly neutralize SARS-CoV-2 variants.

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H H nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of ≥5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: Dromedary camel ( Camelus dromedarius ) V H H phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.

Characterization of a Compound Heterozygous SLC2A9 Mutation That Causes Hypouricemia.

Renal hypouricemia is a rare genetic disorder. Hypouricemia can present as renal stones or exercise-induced acute renal failure, but most cases are asymptomatic. Our previous study showed that two recessive variants of SLC22A12 (p.Trp258*, pArg90His) were identified in 90% of the hypouricemia patients from two independent cohorts: the Korean genome and epidemiology study (KoGES) and the Korean Cancer Prevention Study (KCPS-II). In this work, we investigate the genetic causes of hypouricemia in the rest of the 10% of unsolved cases. We found a novel non-synonymous mutation of SLC2A9 (voltage-sensitive uric acid transporter) in the whole-exome sequencing (WES) results. Molecular dynamics prediction suggests that the novel mutation p.Met126Val in SLCA9b (p.Met155Val in SLC2A9a) hinders uric acid transport through a defect of the outward open geometry. Molecular analysis using Xenopus oocytes confirmed that the p.Met126Val mutation significantly reduced uric acid transport but does not affect the SLC2A9 protein expression level. Our results will shed light on a better understanding of SLC2A9-mediated uric acid transport and the development of a uric acid-lowering agent.

CAR T cells targeting tumor-associated exons of glypican 2 regress neuroblastoma in mice.

Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.

TNIK Is a Therapeutic Target in Lung Squamous Cell Carcinoma and Regulates FAK Activation through Merlin.

Lung squamous cell carcinoma (LSCC) is the second most prevalent type of lung cancer. Despite extensive genomic characterization, no targeted therapies are approved for the treatment of LSCC. Distal amplification of the 3q chromosome is the most frequent genomic alteration in LSCC, and there is an urgent need to identify efficacious druggable targets within this amplicon. We identify the protein kinase TNIK as a therapeutic target in LSCC. TNIK is amplified in approximately 50% of LSCC cases. TNIK genetic depletion or pharmacologic inhibition reduces the growth of LSCC cells in vitro and in vivo. In addition, TNIK inhibition showed antitumor activity and increased apoptosis in established LSCC patient-derived xenografts. Mechanistically, we identified the tumor suppressor Merlin/NF2 as a novel TNIK substrate and showed that TNIK and Merlin are required for the activation of focal adhesion kinase. In conclusion, our data identify targeting TNIK as a potential therapeutic strategy in LSCC. SIGNIFICANCE: Targeted therapies have not yet been approved for the treatment of LSCC, due to lack of identification of actionable cancer drivers. We define TNIK catalytic activity as essential for maintaining LSCC viability and validate the antitumor efficacy of TNIK inhibition in preclinical models of LSCC.This article is highlighted in the In This Issue feature, p. 1307.

Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites.

SARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer's cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain's potential permeation pathway has six constrictions narrowing to about 1 Å in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity's electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

Using iCn3D and the World Wide Web for structure-based collaborative research: Analyzing molecular interactions at the root of COVID-19.

The COVID-19 pandemic took us ill-prepared and tackling the many challenges it poses in a timely manner requires world-wide collaboration. Our ability to study the SARS-COV-2 virus and its interactions with its human host in molecular terms efficiently and collaboratively becomes indispensable and mission-critical in the race to develop vaccines, drugs, and neutralizing antibodies. There is already a significant corpus of 3D structures related to SARS and MERS coronaviruses, and the rapid generation of new structures demands the use of efficient tools to expedite the sharing of structural analyses and molecular designs and convey them in their native 3D context in sync with sequence data and annotations. We developed iCn3D (pronounced "I see in 3D")1 to take full advantage of web technologies and allow scientists of different backgrounds to perform and share sequence-structure analyses over the Internet and engage in collaborations through a simple mechanism of exchanging "lifelong" web links (URLs). This approach solves the very old problem of "sharing of molecular scenes" in a reliable and convenient manner. iCn3D links are sharable over the Internet and make data and entire analyses findable, accessible, and reproducible, with various levels of interoperability. Links and underlying data are FAIR2 and can be embedded in preprints and papers, bringing a 3D live and interactive dimension to a world of text and static images used in current publications, eliminating at the same time the need for arcane supplemental materials. This paper exemplifies iCn3D capabilities in visualization, analysis, and sharing of COVID-19 related structures, sequence variability, and molecular interactions.

Author Correction: Safety and feasibility of anti-CD19 CAR T cells with fully human binding domains in patients with B-cell lymphoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Safety and feasibility of anti-CD19 CAR T cells with fully human binding domains in patients with B-cell lymphoma.

Anti-CD19 chimeric antigen receptor (CAR)-expressing T cells are an effective treatment for B-cell lymphoma, but often cause neurologic toxicity. We treated 20 patients with B-cell lymphoma on a phase I, first-in-human clinical trial of T cells expressing the new anti-CD19 CAR Hu19-CD828Z (NCT02659943). The primary objective was to assess safety and feasibility of Hu19-CD828Z T-cell therapy. Secondary objectives included assessments of blood levels of CAR T cells, anti-lymphoma activity, second infusions and immunogenicity. All objectives were met. Fifty-five percent of patients who received Hu19-CD828Z T cells obtained complete remission. Hu19-CD828Z T cells had clinical anti-lymphoma activity similar to that of T cells expressing FMC63-28Z, an anti-CD19 CAR tested previously by our group, which contains murine binding domains and is used in axicabtagene ciloleucel. However, severe neurologic toxicity occurred in only 5% of patients who received Hu19-CD828Z T cells, whereas 50% of patients who received FMC63-28Z T cells experienced this degree of toxicity (P = 0.0017). T cells expressing Hu19-CD828Z released lower levels of cytokines than T cells expressing FMC63-28Z. Lower levels of cytokines were detected in blood from patients who received Hu19-CD828Z T cells than in blood from those who received FMC63-28Z T cells, which could explain the lower level of neurologic toxicity associated with Hu19-CD828Z. Levels of cytokines released by CAR-expressing T cells particularly depended on the hinge and transmembrane domains included in the CAR design.

Electrostatically Driven Guanidinium Interaction Domains that Control Hydrogel-Mediated Protein Delivery In Vivo.

Protein biologics are an important class of drugs, but the necessity for frequent parenteral administration is a major limitation. Drug-delivery materials offer a potential solution, but protein-material adsorption can cause denaturation, which reduces their effectiveness. Here, we describe a new protein delivery platform that limits direct contact between globular protein domains and material matrix, yet from a single subcutaneous administration can be tuned for long-term drug release. The strategy utilizes complementary electrostatic interactions made between a suite of designed interaction domains (IDs), installed onto the terminus of a protein of interest, and a negatively charged self-assembled fibrillar hydrogel. These intermolecular interactions can be easily modulated by choice of ID to control material interaction and desorption energies, which allows regulation of protein release kinetics to fit desired release profiles. Molecular dynamics studies provided a molecular-level understanding of the mechanisms that govern release and identified optimal binding zones on the gel fibrils that facilitate strong ID-material interactions, which are crucial for sustained release of protein. This delivery platform can be easily loaded with cargo, is shear-thin syringe implantable, provides improved protein stability, is capable of a diverse range of in vitro release rates, and most importantly, can accomplish long-term control over in vivo protein delivery.

Contribution of SLC22A12 on hypouricemia and its clinical significance for screening purposes.

Differentiating between inherited renal hypouricemia and transient hypouricemic status is challenging. Here, we aimed to describe the genetic background of hypouricemia patients using whole-exome sequencing (WES) and assess the feasibility for genetic diagnosis using two founder variants in primary screening. We selected all cases (N = 31) with extreme hypouricemia (<1.3 mg/dl) from a Korean urban cohort of 179,381 subjects without underlying conditions. WES and corresponding downstream analyses were performed for the discovery of rare causal variants for hypouricemia. Two known recessive variants within SLC22A12 (p.Trp258*, pArg90His) were identified in 24 out of 31 subjects (77.4%). In an independent cohort, we identified 50 individuals with hypouricemia and genotyped the p.Trp258* and p.Arg90His variants; 47 of the 50 (94%) hypouricemia cases were explained by only two mutations. Four novel coding variants in SLC22A12, p.Asn136Lys, p.Thr225Lys, p.Arg284Gln, and p.Glu429Lys, were additionally identified. In silico studies predict these as pathogenic variants. This is the first study to show the value of genetic diagnostic screening for hypouricemia in the clinical setting. Screening of just two ethnic-specific variants (p.Trp258* and p.Arg90His) identified 87.7% (71/81) of Korean patients with monogenic hypouricemia. Early genetic identification of constitutive hypouricemia may prevent acute kidney injury by avoidance of dehydration and excessive exercise.

The upcoming subatomic resolution revolution.

Subatomic resolution macromolecular crystallography has been revealing the most fascinating details of macromolecular structures for many years. This most extreme form of macromolecular crystallography is going through rapid changes. A new generation of superbrilliant X-ray sources and detectors is facilitating the rapid acquisition of high-quality datasets. Equally important, a new breed of methods and highly integrated advanced computational tools for structure refinement and analysis is poised to change the way we use subatomic resolution data and reposition high-resolution macromolecular crystallography in medicinal chemistry studies. Subatomic resolution macromolecular crystallography may soon be a routine source of detailed molecular information besides precise geometries, including binding energies and other chemical descriptors, opening new possibilities of application.

A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death.

Intracellular lipopolysaccharide (LPS) triggers the non-canonical inflammasome pathway, resulting in pyroptosis of innate immune cells. In addition to its well-known proinflammatory role, LPS can directly cause regression of some tumors, although the underlying mechanism has remained unknown. Here we show that secretoglobin(SCGB)3A2, a small protein predominantly secreted in airways, chaperones LPS to the cytosol through the cell surface receptor syndecan-1; this leads to pyroptotic cell death driven by caspase-11. SCGB3A2 and LPS co-treatment significantly induced pyroptosis of macrophage RAW264.7 cells and decreased cancer cell proliferation in vitro, while SCGB3A2 treatment resulted in reduced progression of xenograft tumors in mice. These data suggest a conserved function for SCGB3A2 in the innate immune system and cancer cells. These findings demonstrate a critical role for SCGB3A2 as an LPS delivery vehicle; they reveal one mechanism whereby LPS enters innate immune cells leading to pyroptosis, and they clarify the direct effect of LPS on cancer cells.

Conformational energy range of ligands in protein crystal structures: The difficult quest for accurate understanding.

In this review, we address a fundamental question: What is the range of conformational energies seen in ligands in protein-ligand crystal structures? This value is important biophysically, for better understanding the protein-ligand binding process; and practically, for providing a parameter to be used in many computational drug design methods such as docking and pharmacophore searches. We synthesize a selection of previously reported conflicting results from computational studies of this issue and conclude that high ligand conformational energies really are present in some crystal structures. The main source of disagreement between different analyses appears to be due to divergent treatments of electrostatics and solvation. At the same time, however, for many ligands, a high conformational energy is in error, due to either crystal structure inaccuracies or incorrect determination of the reference state. Aside from simple chemistry mistakes, we argue that crystal structure error may mainly be because of the heuristic weighting of ligand stereochemical restraints relative to the fit of the structure to the electron density. This problem cannot be fixed with improvements to electron density fitting or with simple ligand geometry checks, though better metrics are needed for evaluating ligand and binding site chemistry in addition to geometry during structure refinement. The ultimate solution for accurately determining ligand conformational energies lies in ultrahigh-resolution crystal structures that can be refined without restraints.

New insights into the enzymatic mechanism of human chitotriosidase (CHIT1) catalytic domain by atomic resolution X-ray diffraction and hybrid QM/MM.

Chitotriosidase (CHIT1) is a human chitinase belonging to the highly conserved glycosyl hydrolase family 18 (GH18). GH18 enzymes hydrolyze chitin, an N-acetylglucosamine polymer synthesized by lower organisms for structural purposes. Recently, CHIT1 has attracted attention owing to its upregulation in immune-system disorders and as a marker of Gaucher disease. The 39 kDa catalytic domain shows a conserved cluster of three acidic residues, Glu140, Asp138 and Asp136, involved in the hydrolysis reaction. Under an excess concentration of substrate, CHIT1 and other homologues perform an additional activity, transglycosylation. To understand the catalytic mechanism of GH18 chitinases and the dual enzymatic activity, the structure and mechanism of CHIT1 were analyzed in detail. The resolution of the crystals of the catalytic domain was improved from 1.65 Š(PDB entry 1waw) to 0.95-1.10 Šfor the apo and pseudo-apo forms and the complex with chitobiose, allowing the determination of the protonation states within the active site. This information was extended by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. The results suggest a new mechanism involving changes in the conformation and protonation state of the catalytic triad, as well as a new role for Tyr27, providing new insights into the hydrolysis and transglycosylation activities.