Expression, Assay, and Structure of the Extracellular Domain of Murine Carbonic Anhydrase XIV
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m310809200
ISSN1083-351X
AutoresDouglas A. Whittingtons, Jeffrey H. Grubb, Abdül Waheed, Gul N. Shah, William S. Sly, D.W. Christianson,
Tópico(s)Chemical Reactions and Isotopes
ResumoCarbonic anhydrase (CA) XIV is the most recently identified mammalian carbonic anhydrase isozyme, and its presence has been demonstrated in a number of tissues. Full-length CA XIV is a transmembrane protein composed of an extracellular catalytic domain, a single transmembrane helix, and a short intracellular polypeptide segment. The amino acid sequence identity of human CA XIV relative to the other membrane-associated isozymes (CA IV, CA IX, and CA XII) is 34-46%. We report here the expression and purification of both the full-length enzyme and a truncated, secretory form of murine CA XIV. Both forms of this isozyme are highly active, and both show an abrogation of activity in the presence of 0.2% SDS, in contrast to the behavior of murine CA IV. We also report the crystal structure of the extracellular domain of murine CA XIV at 2.8 Å resolution and of an enzyme-acetazolamide complex at 2.9 Å resolution. The structure shows a monomeric glycoprotein with a topology similar to that of other mammalian CA isozymes. Based on the x-ray crystallographic results, we compare and contrast known structures of membrane-associated CA isozymes to rationalize the structural elements responsible for the SDS resistance of CA IV and to discuss prospects for the design of selective inhibitors of membrane-associated CA isozymes. Carbonic anhydrase (CA) XIV is the most recently identified mammalian carbonic anhydrase isozyme, and its presence has been demonstrated in a number of tissues. Full-length CA XIV is a transmembrane protein composed of an extracellular catalytic domain, a single transmembrane helix, and a short intracellular polypeptide segment. The amino acid sequence identity of human CA XIV relative to the other membrane-associated isozymes (CA IV, CA IX, and CA XII) is 34-46%. We report here the expression and purification of both the full-length enzyme and a truncated, secretory form of murine CA XIV. Both forms of this isozyme are highly active, and both show an abrogation of activity in the presence of 0.2% SDS, in contrast to the behavior of murine CA IV. We also report the crystal structure of the extracellular domain of murine CA XIV at 2.8 Å resolution and of an enzyme-acetazolamide complex at 2.9 Å resolution. The structure shows a monomeric glycoprotein with a topology similar to that of other mammalian CA isozymes. Based on the x-ray crystallographic results, we compare and contrast known structures of membrane-associated CA isozymes to rationalize the structural elements responsible for the SDS resistance of CA IV and to discuss prospects for the design of selective inhibitors of membrane-associated CA isozymes. Carbonic anhydrases (CAs) 1The abbreviation used is: CA, carbonic anhydrase. are zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide (CO2+H2O⇔HCO3-+H+). This simple chemical reaction has important implications for pH homeostasis, carbon dioxide and ion transport, respiration, and many other critical processes in living systems (1Parkkila S. Chegwidden W.R. Carter N.D. Edwards Y.H. The Carbonic Anhydrases: New Horizons. Birkhaüser Verlag, Basel, Switzerland2000: 79-93Crossref Scopus (44) Google Scholar). The CAs fall into three distinct classes (α, β, and γ) on the basis of sequence and structural similarity (2Hewett-Emmett D. Chegwidden W.R. Carter N.D. Edwards Y.H. The Carbonic Anhydrases: New Horizons. Birkhaüser Verlag, Basel, Switzerland2000: 29-76Crossref Scopus (105) Google Scholar). All mammalian CAs belong to the α class, of which there are at least 11 enzymatically active isozymes. Additional CA-related proteins lacking intact zinc-binding sites have been identified based on amino acid sequence identity to active isozymes (3Tashian R.E. Hewett-Emmett D. Carter N. Bergenhem N.C.H. Chegwidden W.R. Carter N.D. Edwards Y.H. The Carbonic Anhydrases: New Horizons. Birkhäuser Verlag, Basel, Switzerland2000: 105-120Crossref Scopus (60) Google Scholar). The CAs are ubiquitous in mammalian tissues, but individual isozymes display tissue-specific distributions (1Parkkila S. Chegwidden W.R. Carter N.D. Edwards Y.H. The Carbonic Anhydrases: New Horizons. Birkhaüser Verlag, Basel, Switzerland2000: 79-93Crossref Scopus (44) Google Scholar). Further distinctions in isozyme localization are due to the cytosolic, membrane-associated, or secretory nature of specific CAs. The varying tissue distributions have been exploited for the development of CA inhibitors targeted to specific regions of the body, the most notable example being topically applied compounds such as dorzolamide and brinzolamide for the treatment of glaucoma (4Sugrue M.F. Prog. Retinal Eye Res. 2000; 19: 87-112Crossref PubMed Scopus (209) Google Scholar). Subtle structural differences among the CA isozymes also hold promise for the development of isozyme-specific inhibitors, certain examples of which have been demonstrated (5Supuran C.T. Scozzafava A. Casini A. Med. Res. Rev. 2003; 23: 146-189Crossref PubMed Scopus (1165) Google Scholar). The most recently identified mammalian CA isozyme is CA XIV. Through the use of Northern blotting and reverse transcriptase-polymerase chain reaction techniques, CA XIV mRNA has been demonstrated in kidney, liver, brain, skeletal muscle, heart, and lung (6Mori K. Ogawa Y. Ebihara K. Tamura N. Tashiro K. Kuwahara T. Mukoyama M. Sugawara A. Ozaki S. Tanaka I. Nakao K. J. Biol. Chem. 1999; 274: 15701-15705Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 7Fujikawa-Adachi K. Nishimori I. Taguchi T. Onishi S. Genomics. 1999; 61: 74-81Crossref PubMed Scopus (112) Google Scholar, 8Kaunisto K. Parkkila S. Rajaniemi H. Waheed A. Grubb J. Sly W.S. Kidney Int. 2002; 61: 2111-2118Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The protein itself has been identified in murine and human brain, murine liver, and rat and murine kidney (8Kaunisto K. Parkkila S. Rajaniemi H. Waheed A. Grubb J. Sly W.S. Kidney Int. 2002; 61: 2111-2118Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 9Parkkila S. Parkkila A.-K. Rajaniemi H. Shah G.N. Grubb J.H. Waheed A. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1918-1923Crossref PubMed Scopus (112) Google Scholar, 10Parkkila S. Kivelä A.J. Kaunisto K. Parkkila A.-K. Hakkola J. Rajaniemi H. Waheed A. Sly W.S. BMC Gastroenterol. 2002; 2: 1-7Crossref PubMed Scopus (38) Google Scholar). CA XIV is a bitopic membrane protein with an extracellular N-terminal catalytic domain, a single membrane-spanning segment, and a small intracellular C-terminal polypeptide containing potential phosphorylation sites (6Mori K. Ogawa Y. Ebihara K. Tamura N. Tashiro K. Kuwahara T. Mukoyama M. Sugawara A. Ozaki S. Tanaka I. Nakao K. J. Biol. Chem. 1999; 274: 15701-15705Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 7Fujikawa-Adachi K. Nishimori I. Taguchi T. Onishi S. Genomics. 1999; 61: 74-81Crossref PubMed Scopus (112) Google Scholar). The first 15 amino acids are hydrophobic and constitute a signal sequence, and the catalytic domain contains one putative N-glycosylation site (6Mori K. Ogawa Y. Ebihara K. Tamura N. Tashiro K. Kuwahara T. Mukoyama M. Sugawara A. Ozaki S. Tanaka I. Nakao K. J. Biol. Chem. 1999; 274: 15701-15705Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 7Fujikawa-Adachi K. Nishimori I. Taguchi T. Onishi S. Genomics. 1999; 61: 74-81Crossref PubMed Scopus (112) Google Scholar). This topology is similar to that of the other transmembrane isozymes, CA IX and CA XII. A fourth isozyme, CA IV, is also membrane-associated, but the post-translational attachment of a glycosylphosphatidylinositol group to the C terminus of CA IV serves as the membrane anchor rather than the polypeptide itself (11Zhu X.L. Sly W.S. J. Biol. Chem. 1990; 265: 8795-8801Abstract Full Text PDF PubMed Google Scholar). The amino acid sequence identity of human CA XIV relative to the other three membrane-bound CA isozymes is 34-46%. CA XIV also shares 38% sequence identity with CA VI, an extracellular, secreted isozyme found in saliva. Despite similarities in amino acid sequences and overall topology, the membrane-associated CAs differ in tissue distribution. CA XIV is found in regions of liver cells distinct from the location of CA IV (10Parkkila S. Kivelä A.J. Kaunisto K. Parkkila A.-K. Hakkola J. Rajaniemi H. Waheed A. Sly W.S. BMC Gastroenterol. 2002; 2: 1-7Crossref PubMed Scopus (38) Google Scholar), although certain regions of the kidney show positive immunostaining for both of these isozymes, suggesting redundant function (8Kaunisto K. Parkkila S. Rajaniemi H. Waheed A. Grubb J. Sly W.S. Kidney Int. 2002; 61: 2111-2118Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Intriguingly, the presence of an extracellular carbonic anhydrase has long been suspected in mammalian brain (12Walz W. Can. J. Physiol. Pharmacol. 1989; 67: 577-581Crossref PubMed Scopus (43) Google Scholar, 13Chen J.C.T. Chesler M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7786-7790Crossref PubMed Scopus (96) Google Scholar). Known CA inhibitors, including compounds that are impermeable to cells, were shown to enhance the extracellular alkaline shift observed in slices of hippocampus after synaptic transmission (12Walz W. Can. J. Physiol. Pharmacol. 1989; 67: 577-581Crossref PubMed Scopus (43) Google Scholar, 13Chen J.C.T. Chesler M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7786-7790Crossref PubMed Scopus (96) Google Scholar, 14Tong C.-K. Cammer W. Chesler M. Glia. 2000; 31: 125-130Crossref PubMed Scopus (23) Google Scholar). Recent immunostaining results identify CA XIV on neurons and axons in both mouse and human brain, suggesting that this isozyme is responsible for modulating pH shifts during excitatory synaptic transmission (9Parkkila S. Parkkila A.-K. Rajaniemi H. Shah G.N. Grubb J.H. Waheed A. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1918-1923Crossref PubMed Scopus (112) Google Scholar). The other two transmembrane isozymes, CA IX and CA XII, show a varied tissue distribution, but both are overexpressed in certain cancers, and their transcription is regulated by the von Hippel-Landau tumor suppressor (15McKiernan J.M. Buttyan R. Bander N.H. Stifelman M.D. Katz A.E. Chen M.-W. Olsson C.A. Sawczuk I.S. Cancer Res. 1997; 57: 2362-2365PubMed Google Scholar, 16Pastoreková S. Parkkila S. Parkkila A.-K. Opavsky R. Zelník V. Saarnio J. Pastorek J. Gastroenterology. 1997; 112: 398-408Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 17Saarnio J. Parkkila S. Parkkila A.-K. Haukipuro K. Pastoreková S. Pastorek J. Kairaluoma M.I. Karttunen T.J. Am. J. Pathol. 1998; 153: 279-285Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 18Torczynski, R. M., and Bollon, A. P. (December 31, 1996) U. S. Patent 5,589,579Google Scholar, 19Türeci O. Sahin U. Vollmar E. Siemer S. Göttert E. Seitz G. Parkkila A.-K. Shah G.N. Grubb J.H. Pfreundschuh M. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7608-7613Crossref PubMed Scopus (323) Google Scholar, 20Kivelä A. Parkkila S. Saarnio J. Karttunen T.J. Kivelä J. Parkkila A.-K. Waheed A. Sly W.S. Grubb J.H. Shah G. Türeci O. Rajaniemi H. Am. J. Pathol. 2000; 156: 577-584Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Karhumaa P. Parkkila S. Türeci O. Waheed A. Grubb J.H. Shah G. Parkkila A.-K. Kaunisto K. Tapanainen J. Sly W.S. Rajaniemi H. Mol. Hum. Reprod. 2000; 6: 68-74Crossref PubMed Scopus (79) Google Scholar, 22Ivanov S.V. Kuzmin I. Wei M.-H. Pack S. Geil L. Johnson B.E. Stanbridge E.J. Lerman M.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12596-12601Crossref PubMed Scopus (341) Google Scholar). We report here the expression, purification, and assay of the soluble, extracellular domain of murine CA XIV and its structure determination by x-ray crystallographic methods. The x-ray structure confirms that CA XIV is a glycoprotein and helps define its quaternary structure relative to its solution behavior and its similarity to the related CA XII isozyme. The structure of murine CA XIV complexed with acetazolamide is also presented. Based on the structures of CAs IV, XII, and XIV, we rationalize the structural elements responsible for the unique SDS resistance of CA IV. This resistance allowed CA IV to be solubilized from tissues by SDS and purified in the presence of SDS, conditions under which other known CAs were inactive (11Zhu X.L. Sly W.S. J. Biol. Chem. 1990; 265: 8795-8801Abstract Full Text PDF PubMed Google Scholar); subsequently, resistance to SDS became a practical means of determining the contribution of CA IV to total activity in tissues. Finally, prospects for the design of inhibitors selective for the extracellular CA isozymes are discussed based on the structural data. Expression and Purification—The cDNA was cloned by PCR using mRNA from C57BL6 mouse kidney using primers designed by Mori et al. (6Mori K. Ogawa Y. Ebihara K. Tamura N. Tashiro K. Kuwahara T. Mukoyama M. Sugawara A. Ozaki S. Tanaka I. Nakao K. J. Biol. Chem. 1999; 274: 15701-15705Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The sequence 2In this work, the CA XIV sequence was numbered based on alignment with human CA II (see Fig. 3). Inserted residues are numbered by the point of insertion with a letter appended (e.g. 11A, 11B, etc.). differs from that reported for CA XIV from BalbC (6Mori K. Ogawa Y. Ebihara K. Tamura N. Tashiro K. Kuwahara T. Mukoyama M. Sugawara A. Ozaki S. Tanaka I. Nakao K. J. Biol. Chem. 1999; 274: 15701-15705Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and predicts a His instead of a Gln at residue 108 (see Fig. 3) and a 3-base pair in-frame deletion of Ala-263. Mammalian expression vectors containing the cDNA of the wild-type, full-length membrane form and secretory form (I261X) of murine CA XIV were constructed as described (9Parkkila S. Parkkila A.-K. Rajaniemi H. Shah G.N. Grubb J.H. Waheed A. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1918-1923Crossref PubMed Scopus (112) Google Scholar, 23Ulmasov B. Waheed A. Shah G.N. Grubb J.H. Sly W.S. Tu C. Silverman D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14212-14217Crossref PubMed Scopus (57) Google Scholar). Stable Chinese hamster ovary clones expressing the secretory or the full-length membrane form of murine CA XIV were isolated and characterized by CA activity following established procedures (23Ulmasov B. Waheed A. Shah G.N. Grubb J.H. Sly W.S. Tu C. Silverman D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14212-14217Crossref PubMed Scopus (57) Google Scholar). A full-length, membrane form of murine CA XII cDNA was subcloned into the mammalian expression vector pCXN (24Niwa H. Yamamura K.-I. Miyazaki J.-I. Gene (Amst.). 1991; 108: 193-200Crossref PubMed Scopus (4617) Google Scholar) and transiently expressed in COS-7 cells (19Türeci O. Sahin U. Vollmar E. Siemer S. Göttert E. Seitz G. Parkkila A.-K. Shah G.N. Grubb J.H. Pfreundschuh M. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7608-7613Crossref PubMed Scopus (323) Google Scholar). The enzyme expression was analyzed by CA activity measurements (25Sundaram V. Rumbolo P. Grubb J.H. Strisciuglio P. Sly W.S. Am. J. Hum. Genet. 1986; 38: 125-136PubMed Google Scholar). The I261X secretory form of murine CA XIV, designated CA14x, was purified from secretion medium using CA inhibitor affinity chromatography (11Zhu X.L. Sly W.S. J. Biol. Chem. 1990; 265: 8795-8801Abstract Full Text PDF PubMed Google Scholar). The secretion medium was applied to a CA inhibitor affinity column equilibrated with 10 mm HEPES (pH 7.5). The unbound protein was removed by washing with equilibration buffer containing 0 mm and then 150 mm NaCl. The bound enzyme was eluted with 0.1 m sodium acetate containing 0.5 m sodium perchlorate (pH 5.5). The eluted enzyme was concentrated and dialyzed against 10 mm Tris-SO4 (pH 7.5). The homogeneity of the enzyme was assessed by size exclusion chromatography using Sephacryl S-300, SDS-PAGE, and specific activity (25Sundaram V. Rumbolo P. Grubb J.H. Strisciuglio P. Sly W.S. Am. J. Hum. Genet. 1986; 38: 125-136PubMed Google Scholar). Activity Assays—CA activity was measured by the procedure of Maren (26Maren T.H. J. Pharmacol. Exp. Ther. 1960; 130: 26-29PubMed Google Scholar), as described (25Sundaram V. Rumbolo P. Grubb J.H. Strisciuglio P. Sly W.S. Am. J. Hum. Genet. 1986; 38: 125-136PubMed Google Scholar). SDS-resistant CA activity was determined on affinity pure CA samples or membrane-bound CA samples preincubated with 0.2% SDS at room temperature for 30 min prior to activity measurements. The protein concentration was determined by the micro Lowry procedure (27Peterson G.L. Anal. Biochem. 1979; 100: 201-220Crossref PubMed Scopus (886) Google Scholar). CA activity is expressed in enzyme units/mg of cell protein for unpurified enzyme or in enzyme units/mg of affinity pure CA. Crystallization and Data Collection—The CA14x protein was crystallized at room temperature by the hanging drop vapor diffusion method. Drops containing 1.8 μl of 7 mg/ml enzyme in 20 mm sodium phosphate (pH 7.2) and 150 mm NaCl were mixed with 1.8 μl of precipitant buffer (5.5% (w/v) polyethylene glycol 4000, 0.1 m sodium acetate, pH 4.8, 20 mm NaCl) and equilibrated over a well containing 1.0 ml of precipitant buffer. Crystals appeared in the drops within 48 h and grew as long, thin rods to maximum dimensions of 0.7 × 0.03 × 0.03 mm3. For data collection, a microspatula was used to break the rods into shorter pieces that were subsequently harvested into a stabilizing buffer containing 10% (w/v) polyethylene glycol 4000, 0.1 m sodium acetate (pH 4.8), 20 mm NaCl, and 10% (v/v) glycerol. After sequential transfers to stabilizing solutions containing 15 and 25% (v/v) glycerol, the crystals were flash cooled in liquid nitrogen. Diffraction data to 2.8 Å resolution were collected from a single CA14x crystal at beamline X25 of the National Synchrotron Light Source at the Brookhaven National Laboratories. The data were processed with the HKL suite (28Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar) and TRUNCATE (29Collaborative Computational Project No 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The crystals belonged to space group P21 with unit cell dimensions a = 59.0 Å, b = 75.6 Å, c = 73.2 Å, and β = 98.9°; with two molecules in the asymmetric unit, the Matthew's coefficient VM = 2.65 Å3/Da (53% solvent content). For preparation of the CA14x-acetazolamide complex, the crystals were soaked in a stabilizing solution containing 10% (w/v) polyethylene glycol 4000, 22% (v/v) glycerol, 0.1 m sodium acetate (pH 5.5), 20 mm NaCl, and 5 mm acetazolamide for 90 h prior to flash cooling in liquid nitrogen. The data were collected at beamline X12C of National Synchrotron Light Source and processed as described. The data collection statistics are recorded in Table I.Table IData collection and refinement statisticsNative CA14xCA14x and acetazolamideData collectionTotal reflections44,40443,746Unique reflections14,64313,548Resolution range (Å)30−2.8030−2.90Completeness (%)93.8 (94.3)94.0 (68.1)I/σ(I)8.3 (2.5)8.3 (2.5)RmergeaRmerge=∑|I-〈I〉|/∑I, where I is the observed intensity and 〈I〉 is the average intensity calculated for replicate data.0.099 (0.312)0.085 (0.274)RefinementReflections used for refinement13,93313,535Data cut-off (σ)0.00.0RcrystbRcryst=∑||Fo|-|Fc||/∑|Fo|, where|Fo| and|Fc| are the observed and calculated structure factor amplitudes, respectively.0.2340.207RfreecRfree is calculated in the same manner as Rcryst for 9% of reflections excluded from refinement.0.2740.253No. of atomsProtein41144114Solvent9032Carbohydrate6778Average B factor (Å2)4154Root mean square deviationsBonds (Å)0.0060.007Angles (°)1.41.4Dihedrals (°)25.224.5Impropers (°)0.91.0a Rmerge=∑|I-〈I〉|/∑I, where I is the observed intensity and 〈I〉 is the average intensity calculated for replicate data.b Rcryst=∑||Fo|-|Fc||/∑|Fo|, where|Fo| and|Fc| are the observed and calculated structure factor amplitudes, respectively.c Rfree is calculated in the same manner as Rcryst for 9% of reflections excluded from refinement. Open table in a new tab Structure Determination and Refinement—The molecular replacement calculations were performed with AMoRe (30Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) using the atomic coordinates of CA XII (Protein Data Bank code 1JCZ) as a search probe (31Whittington D.A. Waheed A. Ulmasov B. Shah G.N. Grubb J.H. Sly W.S. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9545-9550Crossref PubMed Scopus (249) Google Scholar). Rotation searches using diffraction data from 15-3.5 Å resolution yielded two clear solutions, as ranked by correlation coefficient. Subsequent translation searches placed two molecules in the asymmetric unit, and rigid body refinement lowered the R factor to 0.453. Model building was performed with the program O (32Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), and simulated annealing, conjugate gradient positional refinement, and temperature factor refinements were performed with CNS (33Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Strict noncrystallographic symmetry constraints were applied during initial refinement cycles and were subsequently released into heavily weighted restraints (ω = 300 kcal/mol Å2) for all atoms except those that displayed obvious differences between the two molecules, e.g. due to crystal lattice contacts. The data were refined against a maximum likelihood target function as implemented in CNS, and a bulk solvent correction was employed (ksol = 0.35 e Å-3 was defined) (34Fokine A. Urzhumstev A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1387-1392Crossref PubMed Scopus (38) Google Scholar). Automatic B factor corrections were not used. Solvent molecules were built into the model at positions where the Fo-Fc maps contained peaks of ≥3.0 σ that displayed appropriate hydrogen bonding interactions. The final model had Rcryst = 0.234 (Rfree = 0.274) and included two copies of the CA14x polypeptide, two zinc ions, two acetate ions, four N-acetylglucosamine rings, one mannose ring, and 90 solvent molecules. Geometric parameters were analyzed with PROCHECK (35Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar); a total of 85 and 14% of the backbone ϕ-ψ conformations adopt most favorable and additionally allowed conformations, respectively. The structure of the CA14x-acetazolamide complex was solved using the difference Fourier method starting from the wild-type CA14x structure less all zinc ions, acetate ions, solvent molecules, and sugar moieties. Rigid body, positional, and grouped temperature factor refinements in CNS resulted in a final model having Rcryst = 0.207 (Rfree = 0.253). The refinement protocol was the same as that used for the native CA14x structure, except that an initial B factor correction was applied to the data. The final model contained two CA14x polypeptide chains, two zinc ions, two acetazolamide molecules, four N-acetylglucosamine rings, two mannose rings, and 32 water molecules. The data refinement statistics for both structures are recorded in Table I. Molecular Characterization and Enzyme Activity—Affinity-purified CA14x migrates as a 44-kDa polypeptide on SDS-PAGE (Fig. 1). However, the calculated molecular mass of CA14x deduced from its amino acid sequence is 29.5 kDa. Therefore, the increase in apparent molecular mass of CA14x and the presence of one consensus sequence for N-glycosylation suggest that murine CA XIV is a glycoprotein. Accordingly, the affinity-purified mouse CA14x produced in the glycosylation-defective Lec-1 cell line shows a slightly smaller apparent molecular mass on SDS-PAGE (data not shown). Moreover, CA14x eluted as single peak of 120-kDa mass during size exclusion chromatography on Sephacryl S-300. Because the mass of the monomeric glycopeptide estimated by SDS-PAGE was 44 kDa, these results suggest either that native CA14x exists as a multimer in solution or that its migration on an S-300 column deviates from that of an ideal globular protein. X-ray crystallographic results support the latter explanation (see below). The specific activity of pure recombinant glycosylated CA14x produced in Chinese hamster ovary cells was compared with unglycosylated murine CA II and glycosylated murine CAs IV and XII. The results are presented in Table II. The specific activity of CA XIV (3284 enzyme units/mg) is higher than any of the other isozymes investigated, including CA II. Unlike murine CA IV, CA14x and the secretory form of CA XII were SDS-sensitive. Membrane-associated CA XII and CA XIV were also SDS-sensitive, indicating that CA IV is more stable than even the wild-type transmembrane isozymes. Because CA IV, CA XII, and CA XIV share a common disulfide bond between Cys-23 and Cys-203, the increased stability of CA IV must be ascribed to the additional disulfide bond between Cys-6 and Cys-13 of this isozyme.Table IISpecific activity and SDS resistance of murine carbonic anhydrase isozymesSDS sensitivityaAffinity pure carbonic anhydrase equivalent to 1 enzyme unit, or cell membrane suspension equivalent to 0.5 enzyme unit, was exposed to 0.2% SDS at room temperature for 30 min before activity assay.IsozymeEnzyme units/mg pure CASecretory formMembrane formCA II2500SensitiveCA IVbMurine CA IV has lower activity than murine CA II, as shown, although human CA IV and CA II both have comparable, high level activity (51).436ResistantResistantCA XII155SensitiveSensitiveCA XIV3284SensitiveSensitivea Affinity pure carbonic anhydrase equivalent to 1 enzyme unit, or cell membrane suspension equivalent to 0.5 enzyme unit, was exposed to 0.2% SDS at room temperature for 30 min before activity assay.b Murine CA IV has lower activity than murine CA II, as shown, although human CA IV and CA II both have comparable, high level activity (51Tamai S. Waheed A. Cody L.B. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13647-13652Crossref PubMed Scopus (21) Google Scholar). Open table in a new tab Overall Structure of Murine CA XIV—The structure of the extracellular catalytic domain of murine CA XIV reveals a polypeptide fold characteristic of the α-CA isozymes in which a 10-stranded β-sheet forms the core of the molecule (Fig. 2). A single disulfide linkage is present between residues Cys-23 and Cys-203 that is identical to disulfide bonds found in the membrane-associated isozymes CA IV and CA XII. This disulfide bond helps to stabilize a polypeptide loop in the active site containing Thr-199, a residue that promotes efficient catalysis by orienting the nucleophilic zinc-bound solvent molecule through a hydrogen bonding interaction (36Merz Jr., K.M. J. Mol. Biol. 1990; 214: 799-802Crossref PubMed Scopus (103) Google Scholar, 37Xue Y. Liljas A. Jonsson B.-H. Lindskog S. Proteins Struct. Funct. Genet. 1993; 17: 93-106Crossref PubMed Scopus (69) Google Scholar). Additionally, because CA14x was produced in Chinese hamster ovary cells, the molecule is glycosylated. Both molecules in the asymmetric unit exhibit electron density consistent with N-glycosylation of Asn-195. The CA14x-acetazolamide structure exhibits the highest quality electron density for the carbohydrate. In molecule B, four of the sugar rings that form the core pentasaccharide commonly found in N-glycosylation are visible: two N-acetylglucosamine and two mannose moieties. Molecule A exhibits electron density for only the two N-acetylglucosamines. The catalytic domain of CA XIV exhibits only minor differences compared with the structure of CA XII (31Whittington D.A. Waheed A. Ulmasov B. Shah G.N. Grubb J.H. Sly W.S. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9545-9550Crossref PubMed Scopus (249) Google Scholar), the most closely related transmembrane isozyme. Two loop regions vary between these isozymes; CA XIV has an insert in the Gly-151-Glu-153 loop and a deletion in the Thr-233-Pro-240 loop relative to CA XII. A structure-based sequence alignment is presented in Fig. 3. The catalytic domains of CA XII and CA XIV contain N-glycosylation sites at differing locations in their sequence, but the protein backbones still superimpose well (root mean square deviation = 1.1 Å for 255 Cα atoms). The most striking difference between CA XII and CA XIV is quaternary structure; CA XII is a dimer with 2200 Å2 buried surface area between monomers, whereas CA XIV appears monomeric. The largest surface area buried between adjacent CA XIV molecules in the crystal lattice is 730 Å2 (365 Å2/monomer), which includes the surface area of the carbohydrate, whereas statistical analyses of the buried surface between a large sampling of biological dimers suggests a minimum buried surface area of 1700 Å2 (860 Å2/monomer) (38Ponstingl H. Henrick K. Thornton J.M. Proteins Struct. Funct. Genet. 2000; 41: 47-57Crossref PubMed Scopus (244) Google Scholar). The packing of these two CA14x molecules also occludes the active site of one, further arguing against the existence of a functional CA XIV dimer, and the majority of amino acids present in the human CA XII dimer interface are not conserved in human or murine CA XIV. Even so, the proposed transmembrane α-helix of f
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