We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic Cterminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43-to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using f luorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, ß-glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound ß-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min . Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors .Chloroquine treatment ofnormal human fibroblasts had three effects : (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme . Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect .These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes . This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors .Adsorptive pinocytosis of lysosomal hydrolases was initially recognized as the uptake of "corrective factors" by enzyme-deficient fibroblasts (for review, see reference 14) . These corrective factors J . CELL BIOLOGY C The Rockefeller University Press -0021-9525/80/06/0839/14 $1.00 Volume 85 June 1980 839-852 proved to be acid hydrolases that were secreted into the culture medium by fibroblasts and were also present in body fluids and tissue extracts . The selectivity and saturability of the uptake system 839 on

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 ␣-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 ␣-helical ''130's 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.

The X-ray structure of the homotetrameric lysosomal acid hydrolase, human beta-glucuronidase (332,000 Mr), has been determined at 2.6 A resolution. The tetramer has approximate dihedral symmetry and each promoter consists of three structural domains with topologies similar to a jelly roll barrel, an immunoglobulin constant domain and a TIM barrel respectively. Residues 179-204 form a beta-hairpin motif similar to the putative lysosomal targeting motif of cathepsin D, supporting the view that lysosomal targeting has a structural basis. The active site of the enzyme is formed from a large cleft at the interface of two monomers. Residues Glu 451 and Glu 540 are proposed to be important for catalysis. The structure establishes a framework for understanding mutations that lead to the human genetic disease mucopolysaccharidosis VII, and for using the enzyme in anti-cancer therapy.

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Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of ␤-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate͞insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. 131 I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate (Kin) of 131 I-P-GUS in 2-day-old mice was 0.21 l͞g⅐min and decreased with age. By 7 weeks of age, transport of 131 I-P-GUS was not significant. Capillary depletion revealed that 62% of the 131 I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of 131 I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner. In contrast, the Kin of 131 I-NP-GUS (0.04 l͞g⅐min) was significantly lower than 131 I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither 131 I-P-GUS nor 131 I-NP-GUS crossed the BBB in adult mice. These results indicate that 131 I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate͞insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice.␤-glucuronidase ͉ mannose 6-phosphate͞insulin-like growth factor II receptor ͉ central nervous system ͉ lysosomal storage disease ͉ phosphorylated ␤-glucuronidase

Enzyme replacement therapy (ERT) effectively reverses storage in several lysosomal storage diseases. However, improvement in brain is limited by the blood-brain barrier except in the newborn period. In this study, we asked whether this barrier could be overcome by higher doses of enzyme than are used in conventional trials. We measured the distribution of recombinant human ␤-glucuronidase (hGUS) and reduction in storage by weekly doses of 0.3-40 mg͞kg administered i.v. over 1-13 weeks to mucopolysaccharidosis type VII mice immunotolerant to recombinant hGUS. Mice given up to 5 mg͞kg enzyme weekly over 3 weeks had moderate reduction in meningeal storage but no change in neocortical neurons. Mice given 20 -40 mg͞kg three times over 1 week showed no reduction in storage in any area of the CNS except the meninges. In contrast, mice receiving 4 mg͞kg per week for 13 weeks showed clearance not only in meninges but also in parietal neocortical and hippocampal neurons and glia. Mice given 20 mg͞kg once weekly for 4 weeks also had decreased neuronal, glial, and meningeal storage and averaged 2.5% of wild-type hGUS activity in brain. These results indicate that therapeutic enzyme can be delivered across the blood-brain barrier in the adult mucopolysaccharidosis type VII mouse if administered at higher doses than are used in conventional ERT trials and if the larger dose of enzyme is administered over a sufficient period. These results may have important implications for ERT for lysosomal storage diseases with CNS involvement.␤-glucuronidase deficiency ͉ immune tolerance ͉ lysosomal storage disease ͉ mannose-6-phosphate receptor T he mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases (LSD) caused by the deficiency of enzymes needed for the stepwise degradation of glycosaminoglycans (GAGs). The widespread lysosomal accumulation of undegraded GAGs leads to progressive cellular and organ dysfunction (1). Current treatments for patients with MPSs include hematopoietic stem cell transplantation and enzyme replacement therapy (ERT) (2, 3). MPS type VII (also known as Sly disease) results from deficiency of ␤-D-glucuronoside glucuronosohydrolase (GUS; EC 3.2.1.31) and is inherited as an autosomal recessive trait. Affected patients share many clinical features with patients with other MPSs, including shortened life span, mental retardation, organomegaly, and bone and joint abnormalities, that are collectively referred to as dysostosis multiplex (4). The murine model of MPS VII has proven valuable for the evaluation of novel therapies for LSDs, including bone marrow transplantation, neural progenitor cell transplantation, somatic cell gene replacement therapy, and ERT (5).Previous studies have shown that i.v. injection of a fixed-dose of recombinant murine ␤-glucuronidase (mGUS) initiated at birth reduced pathological evidence of disease and prevented some of the learning, memory, and hearing deficits in the MPS VII mouse (6). However, recombinant mGUS reduced lysosomal storage in the neurons of the brain only i...

Carbonic anhydrase isozyme XII is a recently discovered member of the