Gul N. Shah

Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells.

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC 4.2.1.1) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning α-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-Å resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix–helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby α-helical “130s segment.” The structure of the CA XII–acetazolamide complex is also reported at 1.50-Å resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.

Lipopolysaccharide alters the blood–brain barrier transport of amyloid β protein: A mechanism for inflammation in the progression of Alzheimer’s disease

Alzheimers disease (AD) brains are characterized by accumulation of amyloid beta protein (Abeta) and neuroinflammation. Increased blood-to-brain influx and decreased brain-to-blood efflux across the blood-brain barrier (BBB) have been proposed as mechanisms for Abeta accumulation. Epidemiological studies suggest that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the progression of AD. We hypothesized that inflammation alters BBB handling of Abeta. Mice treated with lipopolysaccharide (LPS) had increased brain influx and decreased brain efflux of Abeta, recapitulating the findings in AD. Neither influx nor efflux was mediated by LPS acting directly on BBB cells. Increased influx was mediated by a blood-borne factor, indomethacin-independent, blocked by the triglyceride triolein, and not related to expression of the blood-to-brain transporter of Abeta, RAGE. Serum levels of IL-6, IL-10, IL-13, and MCP-1 mirrored changes in Abeta influx. Decreased efflux was blocked by indomethacin and accompanied by decreased protein expression of the brain-to-blood transporter of Abeta, LRP-1. LPS paradoxically increased expression of neuronal LRP-1, a major source of Abeta. Thus, inflammation potentially increases brain levels of Abeta by three mechanisms: increased influx, decreased efflux, and increased neuronal production.

Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase

The main function of CAs (carbonic anhydrases) is to participate in the regulation of acid-base balance. Although 12 active isoenzymes of this family had already been described, analyses of genomic databases suggested that there still exists another isoenzyme, CA XV. Sequence analyses were performed to identify those species that are likely to have an active form of this enzyme. Eight species had genomic sequences encoding CA XV, in which all the amino acid residues critical for CA activity are present. However, based on the sequence data, it was apparent that CA XV has become a non-processed pseudogene in humans and chimpanzees. RT-PCR (reverse transcriptase PCR) confirmed that humans do not express CA XV. In contrast, RT-PCR and in situ hybridization performed in mice showed positive expression in the kidney, brain and testis. A prediction of the mouse CA XV structure was performed. Phylogenetic analysis showed that mouse CA XV is related to CA IV. Therefore both of these enzymes were expressed in COS-7 cells and studied in parallel experiments. The results showed that CA XV shares several properties with CA IV, i.e. it is a glycosylated glycosylphosphatidylinositol-anchored membrane protein, and it binds CA inhibitor. The catalytic activity of CA XV is low, and the correct formation of disulphide bridges is important for the activity. Both specific and non-specific chaperones increase the production of active enzyme. The results suggest that CA XV is the first member of the alpha-CA gene family that is expressed in several species, but not in humans and chimpanzees.

Expression of a Novel Transmembrane Carbonic Anhydrase Isozyme XII in Normal Human Gut and Colorectal Tumors

Carbonic anhydrase isozyme XII is a recently discovered member of the alpha-carbonic anhydrase gene family with a suggested role in von Hippel-Lindau gene-mediated carcinogenesis. Increased expression of its mRNA has been observed in renal and lung carcinomas. This paper presents the localization of CA XII in the normal human gut and in colorectal tumors. Immunohistochemistry performed using a polyclonal antibody raised against truncated CA XII revealed prominent polarized staining for CA XII in the basolateral plasma membrane of the enterocytes of the normal large intestine, the reaction being most intense in the surface epithelial cuff region. Most colorectal tumors displayed abnormal expression of CA XII; the most dramatic change was observed in the deep parts of the adenomatous mucosa, where the positive immunoreaction clearly increased along with the grade of dysplasia. Adenomas with severe dysplasia and carcinomas showed an equal, diffuse staining pattern. The results indicate region-specific regulation of CA XII expression along the cranial-caudal axis of the human gut, whereas its diffuse expression in the most malignant tumors seems to correlate with their biological behavior.

Age-related changes in the blood-brain barrier.

Aging of the cerebral microcirculation results in significant alteration in the blood-brain barrier (BBB). The barrier function appears to remain intact in older animals, although it may be more susceptible to disruption by external factors (hypertension) and drugs (haloperidol). While overall transport processes do not change with age, aging animals and humans have altered BBB function of select carrier mediated transport systems including the transport of choline, glucose, butyrate and triiodothyronine. These age-related changes are the result of either alteration in the carrier molecules or the physiochemical properties of the cerebral microvessels. At the present time, it is not known whether changes in the BBB contribute to the age-related neurodegenerative diseases or are merely epiphenomena of aging.

GLUT-1 Expression in the Cerebra of Patients with Alzheimer’s Disease

To investigate the molecular basis of reduced GLUT-1 concentration of the blood-brain barrier in patients with Alzheimers disease (AD), the GLUT-1 mass, mRNA content, and structure were studied in eight patients with AD and seven age-matched controls. The results indicate that the 55-kDa GLUT-1 is significantly reduced in AD without a significant change in GLUT-1 mRNA concentrations. Because in some animal models changes in GLUT-1 expression is associated with changes in GLUT-1 mRNA structure, the length of the poly(A) tail of the GLUT-1 mRNA was estimated with a reverse transcription-polymerase chain reaction technique. The length of poly(A) tail of GLUT-1 mRNA in AD subjects was not significantly different from the controls. It is concluded that the AD-related change in GLUT-1 expression is not the result of altered poly(A) length of GLUT-1 mRNA.

Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone

Autosomal dominant familial isolated hypoparathyroidism (AD-FIH) is caused by a Cys → Arg mutation (C18R) in the hydrophobic core of the signal peptide of human preproparathyroid hormone (PPTH). Although this mutation impairs secretion of the hormone, the mechanism by which one mutant allele produces the autosomal-dominant disease is unexplained. Using transfected HEK293 cells, we demonstrate that the expressed mutant hormone is trapped intracellularly, predominantly in the endoplasmic reticulum (ER). This ER retention was found to be toxic for the cells, which underwent apoptosis, as evident from the marked increase in the number of cells staining positive for Annexin V binding and for the TUNEL reaction. The cells producing mutant hormone also had marked up-regulation of the ER stress-responsive proteins, BiP and PERK, as well as the proapoptotic transcription factor, CHOP. Up-regulation of these markers of the unfolded protein response supported a causal link between the ER stress and the cell death cascade. When the C18R PPTH was expressed in the presence of 4-phenylbutyric acid, which is a pharmacological chaperone, intracellular accumulation was reduced and normal secretion was restored. This treatment also produced remarkable reduction of ER stress signals and protection against cell death. These data implicate ER stress-induced cell death as the underlying mechanism for AD-FIH and suggest that the pharmacological manipulation of this pathway by using chemical chaperones offers a therapeutic option for treating this disease.

Topiramate Treatment Protects Blood-Brain Barrier Pericytes from Hyperglycemia-Induced Oxidative Damage in Diabetic Mice

Diabetes mellitus causes cerebral microvasculature deterioration and cognitive decline. The specialized endothelial cells of cerebral microvasculature comprise the blood-brain barrier, and the pericytes (PC) that are in immediate contact with these endothelial cells are vital for blood-brain barrier integrity. In diabetes, increased mitochondrial oxidative stress is implicated as a mechanism for hyperglycemia-induced PC loss as a prerequisite leading to blood-brain barrier disruption. Mitochondrial carbonic anhydrases (CA) regulate the oxidative metabolism of glucose and thus play an important role in the generation of reactive oxygen species and oxidative stress. We hypothesize that the inhibition of mitochondrial CA would reduce mitochondrial oxidative stress, rescue cerebral PC loss caused by diabetes-induced oxidative stress, and preserve blood-brain barrier integrity. We studied the effects of pharmacological inhibition of mitochondrial CA activity on streptozotocin-diabetes-induced oxidative stress and PC loss in the mouse brain. At 3 wk of diabetes, there was significant oxidative stress; the levels of reduced glutathione were lower and those of 3-nitrotyrosine, 4-hydroxy-2-trans-nonenal, and superoxide dismutase were higher. Treatment of diabetic mice with topiramate, a potent mitochondrial CA inhibitor, prevented the oxidative stress caused by 3 wk of diabetes. A significant decline in cerebral PC numbers, at 12 wk of diabetes, was also rescued by topiramate treatment. These results provide the first evidence that inhibition of mitochondrial CA activity reduces diabetes-induced oxidative stress in the mouse brain and rescues cerebral PC dropout. Thus, mitochondrial CA may provide a new therapeutic target for oxidative stress related illnesses of the central nervous system.

Active Site Residues of Human β-Glucuronidase EVIDENCE FOR GLU540 AS THE NUCLEOPHILE AND GLU451 AS THE ACID-BASE RESIDUE

Human β-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves β-d-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coliβ-galactosidase active sites led us to propose that residues Glu451, Glu540, and Tyr504 in hGUSB are involved in catalysis, Glu451 being the acid-base residue and Glu540 the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu451, Glu540, and Tyr504 hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. Thek cat/K m values (mm −1·s−1) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu451 as the acid-base catalyst. We conclude that, like their homologues in E. coli β-galactosidase, Glu540 is the nucleophilic residue, Glu451 the acid-base catalyst, and Tyr504 is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.

Localization of Carbonic Anhydrase XII to the Basolateral Membrane of H -secreting Cells of Mouse and Rat Kidney

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO3 − absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40–45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H+-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO3 − absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.