Transferase-deficiency galactosemia: Immunochemical studies of the Duarte and Los Angeles variants

Summary Rabbit antibodies to purified human placental galactose-l-phosphate uridyltransferase (EC 2.7.7.12) were used to establish immunologic cross-reactivity patterns for the enzyme in hemolysates, prepared from red cells of a normal individual, a homozygous Duarte variant, and a heterozygous Los Angeles variant. The antibody immunoprecipitated all three forms of the enzyme. The amount of antibody absorbed by each hemolysate was related to the different levels of activity, and examination of hemolysate/antibody reaction mixtures by starch gel electrophoresis revealed that the antibody quantitatively precipitated all of the isoenzyme forms that characterize these three genetic variants.This research was supported in part by a United States Public Health Service Grant, HD 06576, from the National Institute of Child Health and Human Development.     

A common mutation associated with the Duarte galactosemia allele.

The human cDNA and gene for galactose-1-phosphate uridyl transferase (GALT) have been cloned and sequenced. A prevalent mutation (Q188R) is known to cause classic galactosemia (G/G). G/G galactosemia has an incidence of 1/38,886 in 1,396,766 Georgia live-born infants, but a more common variant of galactosemia, Duarte, has an unknown incidence. The proposed Duarte biochemical phenotypes of GALT are as follows: D/N, D/D, and D/G, which have approximately 75%, 50%, and 25% of normal GALT activity respectively. In addition, the D allele has isoforms of its enzyme that have more acidic pI than normal. Here we systematically determine (a) the prevalence of an A-to-G transition at base pair 2744 of exon 10 in the GALT gene, transition that produces a codon change converting asparagine to aspartic acid at position 314 (N314D), and (b) the association of this mutation with the Duarte biochemical phenotype. The 2744G nucleotide change adds an AvaII (SinI) cut site, which was identified in PCR-amplified DNA. In 111 biochemically unphenotyped controls with no history of galactosemia, 13 N314D alleles were identified (prevalence 5.9%). In a prospective study, 40 D alleles were biochemically phenotyped, and 40 N314D alleles were found. By contrast, in 36 individuals known not to have the Duarte biochemical phenotype, no N314D alleles were found. We conclude that the N314D mutation is a common allele that probably causes the Duarte GALT biochemical phenotype and occurs in a predominantly Caucasian, nongalactosemic population, with a prevalence of 5.9%.     

Isoelectrofocusing of erythrocyte galactose 1 phospho uridyl transferase in a family with both galactosemia and duarte variants

Summary A family with the presence of the genes for both galactosemia and the Duarte variant is described. Galactose 1 phospho uridyl transferase has been studied not only by electrophoresis on starch gel, but also by isoelectrofocusing on thin-layer acrylamide.Normal and variant transferases were resolved into three bands, the isoelectric point of which was between 5.40 and 5.10 for the normal subjects, and between 5.25 and 4.95 for subjects with the Duarte variant.     

Structure-function relationship in a variant hemoglobin: a combined computational-experimental approach

Our study examines the functional and structural effects of amino acid substitution in the distal side of beta-chains of human Hb Duarte (alpha(2)beta(2)(62Ala-->Pro)). We have compared the functional properties of the purified Hb Duarte with those of HbA, and through proton NMR and molecular dynamics simulations we have investigated their tertiary and quaternary structures. The variant exhibits an increased oxygen affinity with a normal Hill coefficient and Bohr effect. The abnormal function of Hb Duarte is attributed to the presence of a proline residue at the beta62 position, since the functional properties of another Hb variant in the same position, Hb J-Europa (beta(62Ala-->Asp)), have been described as normal. Thereafter (1)H-NMR studies have shown that the beta62 Ala-->Pro substitution causes structural modifications of the tertiary structure of the beta globins, leaving the quaternary structure unaltered. These results have been confirmed by extensive all-atom molecular dynamics simulations. All these findings lead to the conclusion that the beta62 Ala-->Pro substitution produces a destabilization of the E-helix extending downward to the CD corner. Particularly, a cavity near the distal histidine of the beta-chains, connecting the heme pocket to the solvent, is affected, altering the functional properties of the protein molecule.     

Variants of galactose-1-phosphate uridyl transferase in the Greek populations

Summary The frequency of variants of galactose-1-phosphate uridyl transferase was determined among the nine Greek populations by studying a sample of 1570 unselected individuals. Average frequency of normal allele GALT=0.942, galactosemia gene GALT ^G=0.0021 and the Duarte variant gene GALT ^D=0.0548 were observed. Frequency of galactosemia heterozygotes among Greeks was similar to that in other Caucasian populations, but the frequency of the Duarte variant was considerably higher. With the exception of two populations, one with low (Epirus) and one with high (Thrace) frequencies, the polymorphism of the Duarte variant displays very similar frequencies in the various Greek population groups.Supported by Grants HD 01 974 and GM 15 253 from the U.S. Public Health Service     

On the molecular nature of the Duarte variant of galactose-1-phosphate uridyl transferase (GALT)

Galactosemia is an inborn error of galactose metabolism secondary to deficiency of galactose-1-phosphate uridyl transferase (GALT). GALT is a polymorphic enzyme and Duarte (D) is the most common enzyme variant. This variant is characterized by faster electrophoretic mobility and reduced activity. Duarte/galactosemia compound heterozygotes (D/G) are commonly identified in galactosemia newborn screening programs. However, these patients do not generally require treatment. By using a candidate mutation approach to define the molecular basis of the Duarte variant of GALT, a close association between the previously reported N314D polymorphism and the Duarte variant of GALT was found. We suggest that N314D encodes the D variant of GALT and that molecular testing for N314D might be useful to confirm a biochemical diagnosis of Duarte variant of GALT.     

Homozygosity for Pc 1 Duarte-like protein in primates and other animals.

Pc 1 Duarte is a mutant brain protein present in 32% of the normal human population with 2.6% being homozygous. Preliminary studies suggest an increased frequency of this mutation in individuals with affective disease. To determine if this mutant was present in primates, 32 fetal (wild)-born baboons were examined. All 32 were homozygous for a Pc 1 Duarte-like protein.     

Frequency Distribution of Q188R, N314D, Duarte 1, and Duarte 2 GALT Variant Alleles in an Indian Galactosemia Population

Classical galactosemia is a genetic disorder caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. The Q188R and N314D mutations are the most frequently cited GALT gene mutations. N314D is further associated with two variants, Duarte 1 and Duarte 2. Nevertheless, no reports are available on the clinical and molecular spectrum of galactosemia from the Indian population. The present study was designed to establish the frequency of these two most common mutations and their variants in Indian galactosemia patients so as to determine a single most common mutation/polymorphism for establishing the DNA-based diagnosis of galactosemia. Three alleles were found to be present at a frequency of 0.036 (Q188R), 0.40 (N314D), and 0.39 (D2); no D1 alleles were found. A significantly higher frequency of the Duarte 2 allele in our population suggests the presence of a milder form of galactosemia, which can be well managed by early diagnosis and dietary management.     

Characterization of normal and abnormal variants of galactose-1-phosphate uridylyltransferase (EC 2.7.7.12) by isoelectric focusing

Isoelectric focusing (IEF) in polyacrylamide gels has been used to study the isozymes of human galactose-1-phosphate uridylyltransferase (GALT) in erythrocytes and fibroblasts. In addition to the usefulness of IEF in differentiating normal, Duarte variant, and galactosemic homozygotes and heterozygotes, the ability of IEF to distinguish the residual GALT activity in two different galactosemic fibroblast lines and in revertants from them is demonstrated.     

Detection of common mutations in the GALT gene through ARMS

Type I galactosemia is an inborn error resulting from mutations on both alleles of the GALT gene, which leads to the absence or deficiency of galactose-1-phosphate uridyltranseferase (GALT), the second of three enzymes catalyzing the conversion of galactose into glucose. On the basis of residual GALT activity, Type I galactosemia is classified into severe “Classical” and mild “Duarte” phenotypes. Classical galactosemia is frequently associated with S135L, Q188R and K285N mutations in the GALT gene. The functionally neutral N314D variation in the GALT gene is associated with Duarte galactosemia and is widespread among various worldwide populations. The present study aimed at detecting S135L, Q188R and K285N mutations and the N314D variant in the GALT gene by PCR using amplification refractory mutation system (ARMS). ARMS assays were established using standard DNA samples and were used for 8 galactosemia patients and 190 unrelated normal subjects all of Pakistani origin. S135L and K285N mutations were present neither in galactosemia patients nor in normal subjects. Only one galactosemia patient carried Q188R mutation that was in homozygous state. However, the N314D variant was frequently found both in affected (7 out of 16 alleles) and normal subjects (55 out of 380 alleles). This finding indicates that Duarte allele D314 might be far more common in Pakistani population than in European and North American ones.     

A new monoclonal antibody recognizing an antigen of human lymphocytes similar or identical to Tac antigen

A new mouse monoclonal antibody (HIEI, IgG1 type) that reacts with a cell surface glycoprotein of human lymphocytes was isolated. Membrane immunofluorescence assay showed that HIEI, like the anti-Tac monoclonal antibody, reacted preferentially with activated normal human T-cells and adult T-cell leukemia (ATL) virus (ATLV)-carrying human T- and B-cell lines. However, an interesting difference between HIEI and anti-Tac antibody was that HIEI did not react with ATLV-transformed simian cell lines or those cultured with interleukin-2 (IL-2), whereas the anti-Tac antibody did. The immunoprecipitation assay showed that both HIEI and anti-Tac antibody precipitated a glycoprotein with a molecular weight of 60,000 daltons (gp60) from activated normal T-cells and ATLV-positive T- and B-cells, and also gp53 from MT-2 and MT-2-related T-cell lines transformed with ATLV in vitro by the MT-2 cocultivation method. HIEI inhibited the IL-2-dependent proliferation of normal T-cells, but its inhibitory effect was much weaker than that of the anti-Tac antibody. The anti-Tac antibody interfered with the binding of HIEI to target cells, but HIEI did not block binding of the anti-Tac antibody to the cells. These observations indicate that HIEI antibody recognizes a new antigenic determinant of the human Tac antigen.     

Determination of the bifunctional properties of high molecular weight kininogen by studies with monoclonal antibodies directed to each of its chains

In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK). When these proteins are cleaved they are found to have a common heavy chain and bradykinin, but each has a unique light chain. Monoclonal antibodies to the heavy and light chains of HMWK have been developed, and the effects of each on the function of this protein are defined. Initial studies showed that an antibody, C11C1, completely neutralized the coagulant activity of plasma HMWK whereas another antibody, 2B5, did not. On a competitive enzyme-linked immunosorbent assay (CELISA) the C11C1 antibody was consumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma. On immunoblot, the C11C1 antibody recognized one kininogen protein in normal plasma and did not recognize any kininogen antigen in HMWK-deficient plasma. These combined studies indicated that the C11C1 antibody was directed to an epitope on the unique 46-kDa light chain of HMWK. In contrast, the 2B5 antibody on a CELISA was consumed by kininogen antigen in both normal plasma and HMWK-deficient plasma but not by total kininogen-deficient plasma. On immunoblot, the 2B5 antibody recognized both kininogens in normal plasma but only LMWK in HMWK-deficient plasma. These combined studies indicated that the 2B5 antibody was directed to the common 64-kDa heavy chain of the plasma kininogens. Utilizing direct binding studies or competition kinetic experiments, the 2B5 and C11C1 antibodies bound with high affinity (1.71 and 0.77 nM, respectively) to their antigenic determinants on the HMWK molecule. The 2B5 antibody did neutralize the ability of HMWK to inhibit platelet calpain. These studies with monoclonal antibodies directed to each of the HMWK chains indicate that HMWK is a bifunctional molecule that can serve as a cofactor for serine zymogen activation and an inhibitor of cysteine proteases.     

Inhibition of the immune response by membrane-bound antibody.

Cells from the spleens of "normal" swine, which were pretreated with pronase to remove surface membrane-bound immunoglobulin, gave an enhanced hemolytic plaque-forming cell response to sheep red blood cells in vitro in comparison with untreated controls. The enhancement could be abrogated by preincubating pronase-treated spleeen cells in preparations containing antibody to sheep red blood cells. This effect was demonstrated by autologous sera, immune sera, and all three known classes of porcine serum immunoglobulins, including IgM, IgA, and IgG and could be removed by absorption with sheep red blood cells. Surface membrane-bound antibody exerted its effect by binding to the nonadherent cell population. The response of normal spleen cells was unaffected by antibody treatment. Pronase-treatment was not mitogenic, did not function as a polyclonal B cell activator, and did not selectively eliminate T or B cells. The results indicate that removal of antibody from the surface of lymphoid cells enhanced the humoral immune response invitro and confirm that membrane-bound antibody can inhibit response to antigen.     

Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

Murine T lymphoma cells express a novel membrane-associated antigen with unique features

A novel membrane-associated antigen expressed on various murine T lymphoma cells has been detected by a rat monoclonal antibody. The antibody YE6/6 initially produced against Moloney leukemia virus-transformed T lymphoma line MBL-2, reacted with several other lymphoma lines including non-T lymphoma lines as well as thymocytes from leukemic AKR mice, but it did not show significant reactivities with resting or mitogen-activated normal lymphocytes by flow cytometric analysis. The antibody did not bind to some Abelson leukemia-transformed cells, which express Moloney virus antigens, suggesting that the antigen is unlikely to be encoded by Moloney virus genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the antigen molecules immunoprecipitated by the antibody revealed three major polypeptides. Two of the polypeptides, with approximate m.w. of 95,000 and 35,000, can be labeled by the cell surface iodination and, therefore, seem to be exposed on the cell surface. The third polypeptide, with approximate m.w. of 65,000, is not labeled by the surface iodination but it is readily detected by [35S]methionine labeling. The third polypeptide was labeled with [32P]orthophosphate indicating that it is a phosphoprotein. Western blot analysis showed that YE6/6 antibody primarily reacts with 35,000 m.w. polypeptide. Furthermore, the same 35,000 m.w. protein was also detected in concanavalin A-activated spleen cells at a low level by Western blot, but normal resting lymphocytes were negative. These results suggest that the antigen detected by YE6/6 antibody may be a cell proliferation-associated antigen and its expression is highly elevated on transformed lymphoma cells as compared to normal mitogen-activated lymphocytes.     

Essential Role of T Cells in the Postexposure Prophylaxis of Rabies in Mice

Athymic nude mice injected intramuscularly with a street strain of rabies virus were not protected against rabies by postexposure administration of beta-propiolactone-inactivated rabies vaccine. In contrast, their normal littermates were completely protected from death by the same vaccination regimens. Nude mice did not produce IgG antibody as a result of the vaccine during the test period of 15 days, whereas normal littermates produced IgG antibody from day 5 after vaccination. However, passive immunization with antirabies hyperimmune mouse ascites showed that antibody was completely ineffective in protecting either nude mice or their normal littermates against rabies when given later than 2 days after infection. No significant difference in the induction of circulating interferon by the vaccination was noted in these mice. Passive transfer of immune spleen cells to nude mice immediately after infection resulted in 30 to 37.5% protection of the mice. Passively transferred spleen cells did not produce detectable amounts of neutralizing antibody in the recipient mice except on day 2 after the transfer, when a low level of antibody was detected. These observations demonstrate the essential role of T cells in the postexposure prophylaxis of rabies in mice. The mechanisms of the failure of postexposure vaccination in nude mice are discussed.     

A new human T-cell differentiation antigen: Unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with extablished leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.PJM is a Junior Faculty Clinical Fellow of the American Cancer Society.     

A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with established leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50 000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.