Structural basis of the recognition of adeno-associated virus by the neurological system-related receptor carbonic anhydrase IV.

Carbonic anhydrase IV (Car4) is a newly identified receptor that allows adeno-associated virus (AAV) 9P31 to cross the blood-brain barrier and achieve efficient infection in the central nervous system (CNS) in mouse models. However, the molecular mechanism by which engineered AAV capsids with 7-mer insertion in the variable region (VR) VIII recognize these novel cellular receptors is unknown. Here we report the cryo-EM structures of AAV9P31 and its complex with Mus musculus Car4 at atomic resolution by utilizing the block-based reconstruction (BBR) method. The structures demonstrated that Car4 binds to the protrusions at 3-fold axes of the capsid. The inserted 7-mer extends into a hydrophobic region near the catalytic center of Car4 to form stable interactions. Mutagenesis studies also identified the key residues in Car4 responsible for the AAV9P31 interaction. These findings provide new insights into the novel receptor recognition mechanism of AAV generated by directed evolution and highlight the application of the BBR method to studying the virus-receptor molecular mechanism.

Necrosis predicts benefit from hypoxia-modifying therapy in patients with high risk bladder cancer enrolled in a phase III randomised trial.

BACKGROUND AND PURPOSE: Addition of carbogen and nicotinamide (hypoxia-modifying agents) to radiotherapy improves the survival of patients with high risk bladder cancer. The study investigated whether histopathological tumour features and putative hypoxia markers predicted benefit from hypoxia modification. MATERIALS AND METHODS: Samples were available from 231 patients with high grade and invasive bladder carcinoma from the BCON phase III trial of radiotherapy (RT) alone or with carbogen and nicotinamide (RT+CON). Histopathological tumour features examined were: necrosis, growth pattern, growing margin, and tumour/stroma ratio. Hypoxia markers carbonic anhydrase-IX and glucose transporter-1 were examined using tissue microarrays. RESULTS: Necrosis was the only independent prognostic indicator (P=0.04). Necrosis also predicted benefit from hypoxia modification. Five-year overall survival was 48% (RT) versus 39% (RT+CON) (P=0.32) in patients without necrosis and 34% (RT) versus 56% (RT+CON) (P=0.004) in patients with necrosis. There was a significant treatment by necrosis strata interaction (P=0.001 adjusted). Necrosis was an independent predictor of benefit from RT+CON versus RT (hazard ratio [HR]: 0.43, 95% CI 0.25-0.73, P=0.002). This trend was not observed when there was no necrosis (HR: 1.64, 95% CI 0.95-2.85, P=0.08). CONCLUSIONS: Necrosis predicts benefit from hypoxia modification in patients with high risk bladder cancer and should be used to select patients; it is simple to identify and easy to incorporate into routine histopathological examination.

Immunohistolocalization and gene expression of the carbonic anhydrase isoenzymes (CA-II and CA-VI) in glands associated with the canine lacrimal apparatus.

Cytosolic and secretory carbonic anhydrase isoenzymes (CA-II and CA-VI, respectively) were detected by immunohistolocalization using specific canine CA-II and CA-VI antisera. CA-II and CA-VI were identified in glands associated with the canine lacrimal apparatus, such as lacrimal gland, superficial gland of the third eyelid (third eyelid gland) and tarsal gland. CA-II and CA-VI mRNA signals were also detected by reverse-transcriptase polymerase chain reaction in the same tissues. Some serous acinar cells and duct segments in the lacrimal gland and serous acinar cells in the third eyelid gland were immunopositive for anti-CA-II and CA-VI antisera. In particular, some immunopositive acini to CA-II and CA-VI on the edge of the third eyelid gland are histologically similar to sebaceous gland cells. Sebaceous gland cells in the tarsal and ciliary glands also showed immunopositivity to both CA antisera. CA-II and CA-VI gene transcripts were detected in the same regions. These results suggest that secreted CA-VI may form together with cytosolic CA-II, a high-activity isozyme mostly considered as a bicarbonate producer, in a mutually complementary system for the maintenance of bicarbonate levels to regulate pH in tear fluid and protect the corneal epithelia against injuries. In sebaceous gland cells in the lacrimal apparatus, CA-VI may be related to lipogenesis in an unknown function.

Basolateral carbonic anhydrase IV in the proximal tubule is a glycosylphosphatidylinositol-anchored protein.

Carbonic anhydrase (CA) IV facilitates HCO(3) reabsorption in the renal proximal tubule by catalyzing the reversible hydration of CO(2). CAIV is tethered to cell membranes via a glycosylphosphatidylinositol (GPI) lipid anchor. As there is basolateral as well as apical CAIV staining in proximal tubule, the molecular identity of basolateral CAIV was examined. Biotinylation of confluent monolayers of rat inner medullary collecting duct cells stably transfected with rabbit CAIV showed apical and basolateral CAIV, and in the cell transfectants expressing high levels of CAIV, a transmembrane form was targeted to the basolateral membrane. Basolateral expression of CAIV ( approximately 46 kDa) was confirmed in normal kidney tissue by Western blotting of vesicle fractions enriched for basolateral membranes by Percoll density fractionation. We examined the mode of membrane linkage of basolaterally expressed CAIV in the kidney cortex. CAIV detected in basolateral or apical membrane vesicles exhibited similar molecular size by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis following deglycosylation, and was equally sensitive to phosphatidylinositol-specific phospholipase C digestion, indicating that CAIV is expressed on the basolateral membrane as a GPI-anchored protein. Half of the hydratase activity of basolateral vesicles was resistant to SDS denaturation, compatible with being CAIV. Thus, GPI-anchored CAIV resides in the basolateral membrane of proximal tubule epithelia where it may facilitate HCO(3) reabsorption via association with kNBC1.

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.

Roles of cytosolic and membrane-bound carbonic anhydrase in renal control of acid-base balance in rainbow trout, Oncorhynchus mykiss.

We tested the hypothesis that cytosolic and membrane-associated carbonic anhydrase (CA IV) are involved in renal urinary acidification and bicarbonate reabsorption in rainbow trout. With the use of homological cloning techniques, a 1,137-bp cDNA was assembled that included an open reading frame encoding for a deduced protein of 297 amino acids. Phylogenetic analysis revealed that this protein was likely a CA IV isoform. With the use of this sequence and a previously described trout cytosolic isoform [tCAc (13)], tools were developed to quantify and localize mRNA and protein for the two CA isoforms. Unlike tCAc, which displayed a broad tissue distribution, trout CA IV mRNA (and to a lesser extent protein) was highly and preferentially expressed in the posterior kidney. The results of in situ hybridization, immunocytochemistry, and standard histological procedures demonstrated that CA IV was likely confined to epithelial cells of the proximal tubule with the protein being expressed on both apical and basolateral membranes. The CA IV-containing tubule cells were enriched with Na(+)-K(+)-ATPase. Similar results were obtained for tCAc except that it appeared to be present in both proximal and distal tubules. The levels of mRNA and protein for tCAc increased significantly during respiratory acidosis (hypercapnia). Although tCA IV mRNA was elevated after 24 h of hypercapnia, tCA IV protein levels were unaltered. By using F3500, a membrane-impermeant (yet filtered) inhibitor of CA, in concert with blood and urine analyses, we demonstrated that CA IV (and possibly other membrane-associated CA isoforms) plays a role in urinary acidification and renal bicarbonate reabsorption.

Expression of carbonic anhydrase IV in rabbit corneal endothelial cells.

OBJECTIVE: To demonstrate the molecular expression of carbonic anhydrase IV (CA IV) in rabbit corneal endothelium. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using cultured and fresh rabbit corneal endothelial total RNA and specific primers for CA IV. The RT-PCR product was subcloned and sequenced. Immunoblotting and indirect immunofluorescence staining were performed to detect protein expression and distribution of CA IV using fresh and cultured rabbit corneal endothelium and rat anti-CA IV polyclonal antibody. RESULTS: RT-PCR screening gave positive bands at the predicted size for CA IV from fresh and cultured rabbit corneal endothelium. Sequencing further confirmed the identity of CA IV in corneal endothelium. Immunoblotting analysis showed a single band at 52 kDa for freshly isolated and cultured endothelial cells. Indirect immunofluorescence staining revealed an apparent positive staining in cultured endothelial cells. CONCLUSION: Carbonic anhydrase IV is expressed in rabbit corneal endothelium, which could contribute to the transendothelial HCO(3)(-) flux that is necessary to maintain corneal hydration and transparency.