Biomedical subjects

A Waheed

Publications and source records attributed to A Waheed.

At least 55 records · Page 3Linked to original sources

PubMed1998

Detection of anti-D following antepartum injections of Rh immune globulin.

Antepartum prophylaxis using Rh immune globulin (RhIG) at 28 weeks of gestation is routine in unsensitized Rh-negative women. As various sources state that anti-D may be detected up to 6 months after administration, we reviewed the medical and laboratory records of all Rh-negative women who delivered at our institution during 1995. For 385 evaluable women, only 137 (35.6%) had anti-D demonstrable in their sera at delivery; 97.8 percent of these delivered within 75 days after administration of RhIG. Of 248 women (64.4%) who delivered in < 76 days after administration of RhIG, 134 (54%) had demonstrable anti-D. For 123 women who delivered between 76 to 95 days after RhIG, only 3 (2.4%) had demonstrable anti-D. Of 14 women who delivered more than 96 days after RhIG, none had anti- D at delivery. These data show that the 300-microg dose used in the United States may not be adequate for antepartum protection and that the detection of anti-D more than 100 days after the administration of RhIG should be viewed with suspicion.

Journal Article↗

PubMed1997

Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common autosomal recessive disease associated with loss of regulation of dietary iron absorption and excessive iron deposition in major organs of the body. Recently, a candidate gene for HH (also called HFE) was identified that encodes a novel MHC class I-like protein. Most patients with HH are homozygous for the same mutation in the HFE gene, resulting in a C282Y change in the HFE protein. Studies in cultured cells show that the C282Y mutation abrogates the binding of the recombinant HFE protein to beta2-microglobulin (beta2M) and disrupts its transport to the cell surface. The HFE protein was shown by immunohistochemistry to be expressed in certain epithelial cells throughout the human alimentary tract and to have a unique localization in the cryptal cells of small intestine, where signals to regulate iron absorption are received from the body. In the studies presented here, we demonstrate by immunohistochemistry that the HFE protein is expressed in human placenta in the apical plasma membrane of the syncytiotrophoblasts, where the transferrin-bound iron is normally transported to the fetus via receptor-mediated endocytosis. Western blot analyses show that the HFE protein is associated with beta2M in placental membranes. Unexpectedly, the transferrin receptor was also found to be associated with the HFE protein/beta2M complex. These studies place the normal HFE protein at the site of contact with the maternal circulation where its association with transferrin receptor raises the possibility that the HFE protein plays some role in determining maternal/fetal iron homeostasis. These findings also raise the question of whether mutations in the HFE gene can disrupt this association and thereby contribute to some forms of neonatal iron overload.

Chromatography, Affinity↗

PubMed1997

Hereditary hemochromatosis: effects of C282Y and H63D mutations on association with beta2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells.

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282-->Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with beta2-microglobulin; a second mutation, His-63-->Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with beta2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

Animals↗

PubMed1997

Immunohistochemistry of HLA-H, the protein defective in patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism that leads to excessive iron storage in the liver and other organs. Recently, between 83 and 100% of HH patients have been found to be homozygous for the same mutation in a novel major histocompatibility complex class I-like gene, called the HLA-H gene. The Cys-282 --> Tyr mutation in HH patients would be expected to disrupt the function of the HLA-H gene product by altering a critical disulfide bridge. As a first step in understanding the function of the HLA-H gene product, we generated an antibody to a C-terminal peptide and used it for immunolocalization of the HLA-H protein in the gastrointestinal tract of Finnish and American subjects presumed not to have HH. Although staining for the HLA-H protein was seen in some epithelial cells in every segment of the alimentary canal, its cellular and subcellular expression in the small intestine were quite distinct from those seen in other segments. In contrast to the stomach and colon, where staining was polarized and restricted to the basolateral surfaces, and in contrast to the epithelial cells of the esophagus and submucosal leukocytes, which showed nonpolarized staining around the entire plasma membrane, the staining in small intestine was mainly intracellular and perinuclear, limited to cells in deep crypts. Prior genetic evidence suggested that a defective HLA-H protein is the molecular basis of HH. Here we show that the HLA-H protein not only varies in its pattern of expression along the cranial/caudal axis of the gastrointestinal tract but that it has a unique subcellular localization in the crypts of the small intestine in proximity to the presumed sites of iron absorption.

Cerebral Cortex↗

PubMed1997

Catalysis and inhibition of human carbonic anhydrase IV.

Carbonic anhydrase IV (CA IV) is a membrane-bound form of carbonic anhydrase. We have characterized the catalytic activity and inhibition of recombinant human CA IV. CA IV is a high-activity isozyme in CO2 hydration with a pH-independent kcat value (1.1 x 10(6) s(-1)) comparable to that of CA II (8 x 10(5) s(-1)). Furthermore, CA IV is more active in HCO3- dehydration than is CA II as illustrated by the nearly 3-fold increase in kcat/K(M) to 3 x 10(7) M(-1) s(-1). However, the esterase activity of CA IV is decreased 150-fold compared to CA II. The catalytic mechanisms of CA II and CA IV are nearly identical. Both isozymes show similar dependence on buffer concentration with the rate-limiting step at high buffer concentration being intramolecular proton transfer, although the intramolecular proton transfer for CA IV is 3 times faster than that observed with CA II. Additional positive charges in the active site of CA IV stabilize anions as indicated by a decreased pKa for the Zn-bound water compared to CA II (6.2 vs 6.9), as well as lower inhibition constants for a variety of anions, including halides, sulfate, formate, acetate, and bicarbonate. CA IV is also activated by low concentrations (<20 mM) of chloride, bromide, and phosphate. Activation by phosphate suggests that the phospholipid anchor may be acting both as an extracellular tether and as a protein activator. Finally, the affinity of CA IV for sulfonamide inhibitors is decreased up to 65-fold compared to CA II as demonstrated by fluorescence titration. The increased bicarbonate activity and altered pH profile are consistent with the proposed physiological role of CA IV in renal bicarbonate reabsorption.

Anions↗

PubMed1997

Human carbonic anhydrase IV: in vitro activation and purification of disulfide-bonded enzyme following expression in Escherichia coli.

Human carbonic anhydrase IV (CA IV) expressed in Escherichia coli was refolded and activated in cell extracts with the help of endogenous periplasmic protein disulfide isomerase, DsbA, in the presence of oxidized glutathione. The refolding and activation were inhibited by bacitracin but not affected by known cofactors or activators of other chaperones. Although the yield of the purified CA IV recovered from cell extracts was maximal when activated at 4 degrees C in the presence of 2 mM oxidized glutathione, the rate of refolding and activation was much more rapid at 25 and 37 degrees C. The enzyme purified from the E. coli cell extracts following activation in vitro showed similar structural stability and functional properties as CA IV purified from secretion medium from a stably transfected CHO cell line. These studies suggest that the soluble truncated form of human CA IV expressed in E. coli, which is disulfide-bonded zinc metalloenzyme, can provide a useful model enzyme for studies of protein folding and enzyme activation in vitro. Furthermore, the procedure described for recovery of CA IV following expression in E. coli may be useful for in vitro activation and subsequent purification of other disulfide-containing proteins.

Bacitracin↗

PubMed1997

A study of the radiobiological effectiveness with track etch detectors.

CR-39 nuclear track detectors have been used in this experiment to study the killing effect of low energy protons on V79-753B Chinese hamster cells and to monitor the proton beam. The estimated surviving fractions and the beam parameters are comparable to those found with conventional electronic devices. The surviving fractions fitted by the linear quadratic model support the idea of single-hit mechanism for the cell killing.

Animals↗

PubMed1997

Effect of carbonic anhydrase inhibition and acetoacetate on anaplerotic pyruvate carboxylase activity in cultured rat astrocytes.

In peripheral tissues, carbonic anhydrase (CA) inhibition secondarily decreases the anaplerotic activity of pyruvate carboxylase activity leading to a decline in citric acid cycle intermediates and glutamate. In view of the important role of pyruvate carboxylase in the brain, we examined the effects of CA inhibition on pyruvate-carboxylase-mediated [14C]CO2 fixation in cultured astrocytes from postnatal rat brains. Incubation with H[14C]O3 led to radiolabeling of metabolites found both in the cells and in the medium. These were separated by ion exchange chromatography for identification. Ethoxyzolamide (ETZ), a sulfonamide CA inhibitor (SCAI) with a heterocyclic side group, caused a 43-73% decrease in cell lysate [alpha-ketoglutarate] and 14C incorporation into major products of pyruvate carboxylation in the cell lysates and cell medium (i.e., released products). Half-maximal inhibition of [14C]CO2 fixation was observed between 1 and 3 x 10(7) M. This is similar to the IC50 value for ETZ inhibition of events in other cells that are thought to be mediated by CA. Inhibition was also observed with trifluormethanesulfonamide, an aliphatic SCAI, providing further evidence that this effect is mediated by CA. Western blot analysis using isozyme-specific antisera indicated that astrocytes contain CA II, a cytosolic isozyme, but CA III, CA IV and CA V could not be detected. This finding is unusual since the effects of SCAIs on pyruvate carboxylation in other tissues have been attributed to inhibition of the intramitochondrial isozyme. CA V. [14C]CO2 fixation was also decreased by lowering media [pyruvate] or by addition of 5 mM acetoacetate. It is hypothesized that SCAIs may inhibit pyruvate carboxylation in astrocytes by limiting the supply of bicarbonate to this enzyme while ketone bodies, by inhibiting glucose oxidation, may limit the supply of pyruvate. Interestingly, both SCAIs and ketogenic diets are used to treat adolescent forms of epilepsy. The possibility that these treatments might ultimately work by affecting anaplerotic pyruvate carboxylase activity in the brain is discussed.

Acetoacetates↗

PubMed1997

Common variable immunodeficiency: diagnosis by absent ABO reverse type.

Many causes for ABO discrepancy between red blood cell and serum testing have been cited in the literature. ABO discrepancy due to weak or absent reverse type is most often seen at the extremes of age. We report a case of ABO discrepancy in a 25-year-old woman who presented for wisdom teeth extraction. Initial serologic workup revealed total absence of isohemagglutinin anti-B in this group A, D+ individual. Further evaluation revealed decreased levels of all classes of immunoglobulins and a medical history significant for multiple episodes of infection. Based on the patient's history and laboratory data, she was diagnosed with common variable immunodeficiency (CVID). CVID can cause ABO discrepancy in an adult patient because of an absent ABO reverse type.

Journal Article↗

PubMed1997

Secretory carbonic anhydrase isoenzyme (CA VI) in human serum.

Carbonic anhydrase VI (CA VI) is a secretory isoenzyme that, by analogy to alpha-amylase, is produced in the salivary glands and delivered into saliva. To determine whether CA VI is transferred into the circulation and is detectable in human serum, we collected blood samples from four healthy subjects at 3-h intervals throughout a 24-h period and measured concentrations of CA VI by a specific time-resolved immunofluorometric assay. All serum samples contained CA VI, the concentrations being approximately 22 times lower in serum than in the corresponding saliva samples. The presence of CA VI in serum was confirmed by Western blotting, which under reducing conditions identified a 42-kDa polypeptide band corresponding to the monomeric CA VI. The described time-resolved immunofluorometric assay for CA VI might be useful to identify or exclude diseases of the salivary glands in the differential diagnosis of patients whose serum amylase concentrations are increased.

Blotting, Western↗

PubMed1996

Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-A resolution.

It has recently been demonstrated that the C-terminal deletion mutant of recombinant human carbonic anhydrase IV (G267X CA IV) converts the normally glycosylphosphatidylinositol-anchored enzyme into a soluble secretory form which has the same catalytic properties as the membrane-associated enzyme purified from human tissues. We have determined the three-dimensional structure of the secretory form of human CA IV by x-ray crystallographic methods to a resolution of 2.8 A. Although the zinc binding site and the hydrophobic substrate binding pocket of CA IV are generally similar to those of other mammalian isozymes, unique structural differences are found elsewhere in the active site. Two disufide linkages, Cys-6-Cys-11G and Cys-23-Cys-203, stabilize the conformation of the N-terminal domain. The latter disulfide additionally stabilizes an active site loop containing a cis-peptide linkage between Pro-201 and Thr-202 (this loop contains catalytic residue Thr-199). On the opposite side of the active site, the Val-131-Asp-136 segment adopts an extended loop conformation instead of an alpha-helix conformation as found in other isozymes. Finally, the C terminus is surrounded by a substantial electropositive surface potential, which is likely to stabilize the interaction of CA IV with the negatively charged phospholipid headgroups of the membrane. These structural features are unique to CA IV and provide a framework for the design of sulfonamide inhibitors selective for this particular isozyme.

Amino Acid Sequence↗

PubMed1996

Gly-63-->Gln substitution adjacent to His-64 in rodent carbonic anhydrase IVs largely explains their reduced activity.

Carbonic anhydrase (CA) IV is a glycosyl-phosphatidylinositol-anchored isozyme expressed on plasma membranes of capillary endothelial cells and certain epithelial cells of the nephron, the colon, and the genitourinary tract. CA IVs purified from bovine and rabbit lungs are high-activity enzymes, like human CA IV, while CA IV from mouse and rat lungs had only 10-20% as much catalytic activity. To explain the molecular basis for these differences in activity, we isolated and characterized the full-length cDNAs for bovine and rabbit CA IVs and compared their sequences to those we previously reported for human, murine, and rat CA IVs. These comparisons led us to postulate that a Gly-63-->Gln substitution adjacent to His-64 in the rodent enzymes accounts for their lower activity. To test this hypothesis, we made the Gly-63-->Gln mutants of bovine and rabbit CA IVs and the Gln-63-->Gly mutant of murine CA IV by site-directed mutagenesis, and compared the activities of mutant and wild-type CA IVs expressed in COS-7 cells. In addition, we produced recombinant cDNAs expressing secretory forms of the Gly-63 and Gln-63 forms of each of the three enzymes and compared the activities of the enzymes purified from transfected COS-7 cell secretions with the activities of CA IVs purified from lungs. These studies demonstrated that Gly-63 is important for the high activity of bovine and rabbit CA IVs, and they showed that the low activity of murine CA IV could be improved by the Gln-63-->Gly substitution. We suggest that the lower activity of the rodent CA IVs can be largely explained by the Gln-63 substitution which reduces the efficiency of proton transfer by the adjacent His-64.

Amino Acid Sequence↗

PubMed1996

Carbonic anhydrase IV: purification of a secretory form of the recombinant human enzyme and identification of the positions and importance of its disulfide bonds.

Chinese hamster ovary cells were stably transfected with the cDNA for human carbonic anhydrase IV that was engineered to encode a secretory form of the normally glycosylphosphatidylinositol-anchored membrane protein. Overexpression was achieved by amplification of the cDNA and its dihydrofolate reductase-containing expression vector by growth in the presence of methotrexate. The 33-kDa secretory form of the enzyme was purified to homogeneity from cellular secretions by inhibitor affinity chromatography. Occasional CA IV preparations contained proteolytic fragments of 18 and 15 kDa held together by disulfide bonds. N-terminal sequencing identified the 18-kDa fragment as the N-terminus and the 15-kDa fragment as the C-terminal portion. The specific activity of the purified enzyme preparations (2587 +/- 149 U/mg protein) was comparable to that of enzyme purified from human tissues. In order to identify the cysteines involved in the two disulfide bonds, enzyme purified following metabolic labeling with [35S]cysteine was subjected to proteolytic cleavage and the N-terminal amino acid sequence determined on the labeled peptides isolated by HPLC. Results indicated that the disulfide bonds in the native enzyme link Cys6 to Cys18 and Cys28 to Cys211. Reduction of the enzyme or reduction followed by alkylation both destroy 70% of the enzyme activity and make the enzyme susceptible to inactivation by denaturants. Furthermore, the loss of activity of reduced enzyme on exposure to denaturants is not recovered on removal of denaturants. By contrast, disulfide-bonded enzyme is not only more resistant to inactivation by denaturants, but any loss of activity is reversed on removal of denaturants. Fluorescence anisotropy measurements provided further evidence that the disulfide-bonded enzyme retains structure in the presence of denaturants. Taken together, these results show that the disulfide bonds contribute significantly both to the retention of structure and of catalytic activity in the presence of denaturants, and to the ability to renature following removal of denaturants.

Amino Acid Sequence↗

PubMed1996

Membrane-bound carbonic anhydrase IV is expressed in the luminal plasma membrane of the human gallbladder epithelium.

Alkaline hepatic bile is acidified in the gallbladder to prevent calcium precipitation and gallstone formation. Because membrane-bound carbonic anhydrase (CA) isoenzyme IV participates with cytoplasmic CA II in the acidification of urine in the kidney, we studied its expression in different regions of the human biliary tract using immunohistochemical techniques. The enzyme was expressed in the apical plasma membrane of the gallbladder epithelial cells and in the endothelium of the subepithelial capillaries. In the liver, some epithelial cells of the large bile ducts showed positive staining. Its presence in the gallbladder epithelium could be confirmed by Western blotting, which showed a single 35-kd polypeptide band, corresponding in molecular weight to the intact enzyme. The majority of the enzyme was phased to Triton X-114 detergent phase. A small amount of 35-kd polypeptide was also seen in the water phase. Smaller proteolytic fragments of the enzyme were not seen, suggesting that the tissue sample was well preserved. The results show that CA IV is expressed in abundance in the human gallbladder epithelium, where it may participate together with cytoplasmic CA II and ion transporters in acidification of the gallbladder bile via bicarbonate reabsorption.

Blotting, Western↗

PubMed1996

Differentiation-dependent expression of CA V and the role of carbonic anhydrase isozymes in pyruvate carboxylation in adipocytes.

The incorporation of radioactivity from 14C-labeled compounds into metabolic intermediates and total lipids was examined in 3T3 adipocytes. The heterocyclic sulfonamide carbonic anhydrase inhibitor (SCAI) 6-ethoxyzolamide (ETZ) caused a decrease (42+/-7% of control, IC50 = 2.2+/-1.1 x 10(-7) M) in the incorporation of [14C] bicarbonate into several Krebs cycle intermediates in 3T3-F442A adipocytes. This decrease in pyruvate carboxylase-mediated [14C] carbon fixation was associated with a reduction in fluorometrically determined [citrate] and [malate]. The ability of ETZ to decrease both the incorporation of radioactivity into and the concentrations of Krebs cycle intermediates was not of sufficient magnitude to lower [ATP], but was associated with a decrease in de novo lipogenesis from [14C]glucose. De novo lipogenesis was also inhibited to a similar extent by trifluormethanesulfonamide, an aliphatic SCAI, which suggests that the effects are mediated by carbonic anhydrase. ETZ did not inhibit de novo lipogenesis from [14C]glutamine (12.38+/-1.068 nmol/mg protein, ETZ; 12.5+/-0.846 nmol/mg protein, DMSO). This suggests that ETZ inhibition of lipogenesis involves an inhibitory effect on pyruvate carboxylase as opposed to acetyl CoA carboxylase, because the incorporation of glutamine into lipids does not involve pyruvate carboxylase. Decreased de novo lipogenesis was also observed by incubating cultures in media that contained 1 mM bicarbonate (atmosphere:100% humidified air) rather than 25 mM bicarbonate (atmosphere: 95% humidified air/5% CO2). This suggests that exogenous CO2/bicarbonate may be required to sustain maximal rates of de novo lipogenesis. Because these results implied that CA V, the mitochondrial isoform of carbonic anhydrase, might be present in adipocytes, CA V levels were measured by immunoblotting. Mitochondrial preparations of adipocytes and liver were found to contain similar concentrations of CA V. Unlike adipocyte CA III, CA V concentrations were not significantly different in lean and obese Zucker rats. However, CA V levels were ninefold higher in differentiated 3T3-F442A adipocytes compared to undifferentiated adipoblasts. Our data indicate that CA V is relatively abundant in adipocyte mitochondria and exhibits differentiation-dependent expression like pyruvate carboxylase and the cytosolic isozymes CA II and CA III. The possible roles of CA II and CA V in pyruvate carboxylation are discussed.

3T3 Cells↗

PubMed1995

Carbonic anhydrase IV: role of removal of C-terminal domain in glycosylphosphatidylinositol anchoring and realization of enzyme activity.

Carbonic anhydrase IV (CA IV) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed on the plasma membrane of specific epithelial and endothelial cells. The human cDNA encodes a 312-amino-acid precursor which includes an NH2-terminal signal sequence (residues -18 to -1) that is removed and a C-terminal hydrophobic domain which is cleaved to permit transfer to the GPI anchor. Using biochemical methods, we established that Ser266 is the site of attachment of the GPI anchor to CA IV from human lung. Based on this result, we constructed missense mutants S266F and G267F and a truncation mutant, G267X, and investigated the role of removal of the C-terminal hydrophobic domain on the synthesis and processing of CA IV in transfected COS cells. The G267F mutation had no effect on CA IV expression. By contrast, the S266F mutation prevented removal of the C-terminal domain and the S266F CA IV was inactive, not GPI-anchored, and not expressed on the cell surface. The G267X C-terminal deletion mutation resulted in secretion of an amount of CA IV severalfold higher than the amounts found in cells transfected with wild type cDNA. These results demonstrate that removal of the C-terminal hydrophobic domain is necessary both for GPI anchoring and for realization of CA IV activity. They further show that bypassing C-terminal processing by deletion of the hydrophobic domain leads to secretion of a fully active CA IV in amounts far greater than those which accumulate in cells expressing the wild type, GPI-anchored CA IV.

Amino Acid Sequence↗

PubMed1995

Localization of the insulin-like growth factor II binding site to amino acids 1508-1566 in repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds insulin-like growth factor II (IGF-II) with high affinity. To localize the IGF-II binding site within the 15 repeating units that form the extracytoplasmic domain of the receptor, purified human M6P/IGF-II receptor was digested with thermolysin, and the fragments were analyzed for their ability to bind 125I-IGF-II in a cross-linking assay. Two IGF-II-binding receptor fragments of 23 and 37 kDa were purified. Sequence analysis revealed that the fragments consist of disulfide connected peptides comprising amino acids 1331-1566 and 1331-1697 of the receptor repeats 9-12. In a second approach we expressed truncated forms of the M6P/IGF-II receptor fused to the C terminus of the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor. Fusion proteins containing M6P/IGF-II receptor repeats 10-15, 10-11, or 11-15 bound IGF-II, whereas a fusion protein containing the single repeat 10 failed to bind. This result indicates that repeat 11 (amino acids 1508-1650) is sufficient for binding of IGF-II. Residues 1508-1566, which are shared by the 23-kDa IGF-II-binding fragment and repeat 11, are proposed to form the IGF-II binding site of the M6P/IGF-II receptor.

Amino Acid Sequence↗

PubMed1995

Partial rescue of human carbonic anhydrase II frameshift mutation by ribosomal frameshift.

A single-base-pair deletion in exon 7 of the human carbonic anhydrase II gene was found to be the molecular defect in a group of independently ascertained, clinically heterogeneous, Hispanic carbonic anhydrase II-deficient patients, all of whom had ancestors from the Caribbean islands. This mutation predicts a +1 frameshift at codon 227 and incorporation of 12 missense amino acids before an early stop codon at position 239 produces a 27-kDa truncated carbonic anhydrase II. Expression of the Hispanic mutant cDNA in bacteria produced predominantly the 27-kDa protein, which was inactive. However, a minor 29-kDa polypeptide species was also produced that had 10% the specific activity of the wild-type enzyme after affinity purification. Amino acid sequencing showed that the 29-kDa mutant protein was produced by two frameshift events: a +1 frameshift at codon 227 due to the single-base deletion and a -1 ribosomal frameshift at codon 237 that restored the original reading frame after 11 missense amino acids were incorporated. Antibody against the 11-amino acid frameshift peptide detected the 29-kDa mutant protein in lysates of transfected COS cells. These results indicate that ribosomal frameshift can partially rescue the human carbonic anhydrase II frameshift mutation and suggest a mechanism whereby a compensatory ribosomal frameshift can ameliorate the consequences of certain frameshift mutations. Whether individual differences in efficiency of ribosomal frameshift contribute to clinical heterogeneity in patients with such mutations deserves further study.

Amino Acid Sequence↗