The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase

The Ashkenazi Jewish population is enriched for carriers of a fatal form of Tay-Sachs disease, an inherited disorder caused by mutations in the alpha-chain of the lysosomal enzyme, beta-hexosaminidase A. Until recently it was presumed that Tay-Sachs patients from this ethnic isolate harbored the same alpha-chain mutation. This was disproved by identification of a splice junction defect in the alpha-chain of an Ashkenazi patient which could be found in only 20-30% of the Ashkenazi carriers tested. In this study we have isolated the alpha-chain gene from an Ashkenazi Jewish patient, GM515, with classic Tay-Sachs disease who was negative for the splice junction defect. Sequence analysis of the promoter region, exon and splice junctions regions, and polyadenylation signal area revealed a 4-base pair insertion in exon 11. This mutation introduces a premature termination signal in exon 11 which results in a deficiency of mRNA in Ashkenazi patients. A dot blot assay was developed to screen patients and heterozygote carriers for the insertion mutation. The lesion was found in approximately 70% of the carriers tested, thereby distinguishing it as the major defect underlying Tay-Sachs disease in the Ashkenazi Jewish population.     

A shortened beta-hexosaminidase alpha-chain in an Italian patient with infantile Tay-Sachs disease.

Fibroblasts derived from a beta-hexosaminidase A (HexA)-deficient infant with clinically classic Tay-Sachs disease synthesized a precursor alpha-chain that was smaller than its normal counterpart. Fibroblasts from the infant''s parents, who were consanguinous, produced both normal and mutant alpha-chains. The size difference, estimated to be 2-3 kilodaltons on the basis of sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, persisted after removal of oligosaccharides with endo-H and is therefore attributable to a shortened polypeptide. The mutant alpha-chain did not undergo the further posttranslational modifications characteristic of its normal counterpart--i.e., synthesis of the mannose phosphate recognition marker, association with the beta-chain to give HexA, and proteolytic conversion to the mature form. Nor was it secreted, even in the presence of NH4Cl. Instead, it disappeared in the course of a 20-h chase. These results suggest that the mutant alpha-chain was trapped in an early biosynthetic compartment, either the endoplasmic reticulum or the cis-Golgi. The mutation appears to be different from all those previously described in patients with clinically classic Tay-Sachs disease.     

The molecular organization of the protein HC-IgA complex (HC-IgA)

Complexes of protein HC and monoclonal IgA1 or IgA2 or polyclonal IgA were isolated from human blood plasma. Dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that all complexes contain three types of chains: two light immunoglobulin chains, one regular IgA alpha-chain, and one chain with Mr = 90,000 carrying both alpha-chain and protein HC epitopes. The complexes were split into Fab alpha and Fc alpha fragments by bacterial IgA proteases. The protein HC epitopes were linked to the Fc fragments. Complexes of protein HC and an alpha-chain devoid of the variable region and the first heavy chain constant domain could also be demonstrated to be present in the blood plasma of a patient with alpha-heavy chain disease. Pepsin digestion of HC-IgA released a fragment containing all the protein HC epitopes and the C-terminal nonapeptide of the IgA alpha-chain. The light immunoglobulin chains, the regular alpha-chain, and the 90,000-Da chain from monoclonal HC-IgA1 were isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis and by repeated gel filtration in dodecyl sulfate-containing buffer. The N-terminal amino acid sequence of the alpha-chain was identical with that of a regular human heavy immunoglobulin chain of subgroup III. Subtractive degradations of the 90,000-Da chain displayed 2 amino acid residues in each position in a pattern suggesting simultaneous degradations of a chain identical with the regular alpha-chain of HC-IgA and of uncomplexed, low molecular weight, protein HC. All the results are compatible with a model for HC-IgA in which a single low molecular weight protein HC polypeptide chain is covalently linked, side by side, to the C-terminal nonapeptide of one of the two alpha-chains of a regular monomeric IgA unit.     

Inhibition of beta(S)-chain dependent polymerization by synergistic complementation of contact site perturbations of alpha-chain: application of semisynthetic chimeric alpha-chains

Mouse alpha(1-30)-horse alpha(31-141) chimeric alpha-chain, a semisynthetic super-inhibitory alpha-chain, inhibits beta(S)-chain dependent polymerization better than both parent alpha-chains. Although contact site sequence differences are absent in the alpha(1-30) region of the chimeric chain, the four sequence differences of the region alpha(17-22) could induce perturbations of the side chains at alpha(16), alpha(20) and alpha(23), the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse alpha(31-141) probably results in the super-inhibitory activity of the chimeric alpha-chain. The inhibitory contact site sequence differences, by themselves, could also exhibit similar synergistic complementation. Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin (LM) mutation [His20(alpha)-->Gln], a contact site sequence difference, engineered into human-horse chimeric alpha-chain has been investigated to map such a synergistic complementation. Gln20(alpha) has little effect on the O(2) affinity of HbS, but in human-horse chimeric alpha-chain it reduces the O(2) affinity slightly. In the chimeric alpha-chain, Gln20(alpha) increased sensitivity of the betabeta cleft for the DPG influence, reflecting a cross-talk between the alpha(1)beta(1) interface and betabeta cleft in this semisynthetic chimeric HbS. In the human alpha-chain frame, the polymerization inhibitory activity of Gln20(alpha) is higher compared with horse alpha(1-30), but lower than mouse alpha(1-30). Gln20(alpha) synergistically complements the inhibitory propensity of horse alpha(31-141). However, the inhibitory activity of LM-horse chimeric alpha-chain is still lower than that of mouse-horse chimeric alpha-chain. Therefore, perturbation of multiple contact sites in the alpha(1-30) region of the mouse-horse chimeric alpha-chain and its linkage with the inhibitory propensity of horse alpha(31-141) has been now invoked to explain the super-inhibitory activity of the chimeric alpha-chain. The 'linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contact sites into alpha-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.     

Antibodies to synthetic peptides as probes of acetylcholine receptor structure

Peptides containing the C-terminus of the alpha-chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide-specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alpha-chain. Trypsinization experiments and the use of the unique C-terminal hexapeptide of the alpha-chain demonstrated that the proposed C-terminus does exist on the mature alpha-chain, and that post-translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha-chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa). Cleavage of the alpha-chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9-10 kDa). No signs of experimental myasthenia gravis were observed in rabbits immunised with C-terminal peptide coupled to carrier protein.     

Impaired proteolytic processing of lysosomal N-acetyl-beta-hexosaminidase in cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from patients suffering with infantile generalized N-acetylneuraminic acid (NeuAc) storage disease accumulate free NeuAc in a population of lysosomes less dense than those observed in normal fibroblasts (1.035 vs. greater than 1.07 mean density), as assessed by the distribution of lysosomal enzyme activities and NeuAc on Percoll gradients after subcellular fractionation. In the present study, normal and affected fibroblasts were labeled with [35S]methionine, and cell homogenates or subcellular fractions from Percoll gradients were immunoprecipitated with polyclonal antibodies to lysosomal N-acetyl-beta-hexosaminidase (Hex); immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. The synthesis and initial processing of Hex polypeptides were comparable in normal and affected fibroblasts, but mature polypeptides were quantitatively localized in "buoyant" lysosomes of affected cells, along with Hex activity; moreover, mature alpha-chain of Hex was approximately 2 kDa larger than that observed in normal cells. The molecular weight difference was apparently due to impaired proteolytic processing of alpha-chain in affected fibroblasts, since treatment of immunoprecipitated alpha-chain from normal and affected cells with neuraminidase and endo-beta-N-acetylglucosaminidase H failed to resolve the molecular weight difference. The impaired processing was observed to be persistent (after a chase of up to 200 h), but had no apparent effect on the turnover or activity of Hex in affected fibroblasts. The observed proteolytic processing defect may be primary or secondary in infantile NeuAc storage disease.     

Unique features of the pre-T-cell receptor alpha-chain: not just a surrogate

The pre-T-cell receptor (pre-TCR) has a crucial role in the normal development of alphabeta T cells. Different views have emerged concerning the structure and function of the pre-TCR. This molecular complex can be viewed as a variant of the alphabeta-TCR in which the pre-TCR alpha-chain that is covalently associated with the TCR beta-chain is a 'surrogate' TCR alpha-chain. Alternatively, the unique structure of the pre-TCR might be associated with a unique function, owing to evolutionary selection of a pre-TCR alpha-chain that has different capabilities from the TCR alpha-chain. As described here, I consider that experimental evidence favours the latter view.     

Transport of the IgE receptor alpha-chain is controlled by a multicomponent intracellular retention signal

The human high affinity IgE receptor (FcepsilonRI) is a central component of the allergic response and is expressed as either a trimeric alphagamma2 or tetrameric alphabetagamma2 complex. It has been previously described that the cytoplasmic domain (CD) of the alpha-chain carries a dilysine motif at positions -3/-7 from the C terminus that functions in intracellular retention prior to assembly with other FcepsilonRI subunits. In this report we have further explored the role of the -3/-7 dilysine signal in controlling steady-state alpha-chain transport by mutational analysis and found little surface expression of a -3/-7 dialanine alpha-chain mutant but significant Golgi localization. We compared the transport properties of a series of alpha-chain cytoplasmic domain truncation mutants and observed that truncation mutants lacking 23 or more C-terminal residues showed a dramatic increase in steady-state transport suggesting a role for the membrane-proximal CD sequence in alpha-chain retention. By performing alanine-scanning mutagenesis we identified a dilysine sequence (Lys(212)-Lys(216)) proximal to the transmembrane domain (TMD) that is important for both alpha-chain cell-surface expression and intracellular stability. Furthermore, co-mutation of the Lys(212)-Lys(216) residues with the -3/-7 dilysine signal produced a dramatic increase in alpha-chain surface expression that was further increased by co-mutation of the lone charged residue (Asp(192)) in the TMD thereby defining three regions that function to regulate alpha-chain transport and in a highly synergistic manner.     

Structure of alpha-polymer from in vitro and in vivo highly cross-linked human fibrin.

Peptides derived from plasmic and cyanogen bromide (CNBr) cleavage of highly cross-linked fibrin were isolated and characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analyses, cyanoethylation, and NH2-terminal analyses. Extended plasmic digestions of human fibrin containing four epsilon-(gamma-glutamyl)lysine cross-links per molecule produced a peptide of alpha-chain origin (Mr congruent to 21,000) which was comprised of a small donor peptide cross-linked to the acceptor site peptide from the middle of the alpha-chain. CNBr cleavage of highly cross-linked in vitro fibrin or of fibrin from a spontaneously formed in vivo arterial embolus produced about three cross-linked species of molecular weights 30,000 to 40,000, each of which contained the largest CNBr fragment (Mr = 29,000) from the alpha-chain. The predominant cross-link-containing CNBr fragments derived their donor group from the near COOH-terminal region of the alpha-chain as judged by difference amino acid compositions and NH2-terminal analyses. Additionally, cross-linked fragments of molecular weights 68,000 to 70,000 which appeared to contain two acceptor site peptides (Mr = 29,000) were detected in minor amounts in the CNBr digests of fibrin formed from whole plasma or from purified, plasminogen-free fibrinogen. No larger polymeric cross-linked CNBr fragment was generated from any of the highly cross-linked fibrin preparations examined. A model for the predominant mode of alpha-chain polymerization is proposed.     

Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from Porphyromonas (Bacteroides) gingivalis.

Complement components C3 and C5 are susceptible to limited proteolysis by an arginine-specific cysteine proteinase isolated from Porphyromonas gingivalis. This bacterium is an anaerobe commonly associated with severe periodontal disease. Infection by P. gingivalis is accompanied by an acute inflammatory response, complete with extensive neutrophil involvement. This prompted us to investigate a possible direct role for complement in periodontitis evoked by P. gingivalis. Exposure of C3 and C5 to the cysteine proteinase at molar ratios between 1:25 and 1:100 (enzyme to substrate ratios) resulted in a time-dependent, limited degradation of each component. C3 was converted in a stepwise manner to C3a-like and C3b-like fragments with evidence of extensive further degradation of the C3a-like portion of the molecule. We were unable to demonstrate C3a activity in the C3 digestion mixtures. C3 degradation appears to involve primarily the alpha-chain. Proteolysis of C5 also progresses in a stepwise manner producing an initial internal cleavage of the alpha-chain to generate 30- and 86-kDa fragments. Further digestion of the 86-kDa amino-terminal fragment of the alpha-chain leads to the release of C5a or a C5a-like fragment that is biologically active for neutrophil activation. The fact that a potent chemotactic factor, i.e. C5a, can be generated from C5 by a proteinase derived from P. gingivalis suggests a recruiting mechanism for attracting neutrophils to the gingival lesion site in periodontal disease.     

Export of the high affinity IgE receptor from the endoplasmic reticulum depends on a glycosylation-mediated quality control mechanism

The high affinity IgE receptor (FcepsilonRI) is a multisubunit complex comprised of either alphagamma(2) or alphabetagamma(2) chains. The cotranslational assembly of the IgE-binding alpha-chain with a dimer of gamma-chains occurs in a highly controlled manner and is proposed to involve masking of a dilysine motif present at the cytoplasmic C terminus of the FcepsilonRI alpha-chain that targets localization of this subunit to the endoplasmic reticulum (ER). Here, we show that ER quality control modulates export from the ER of newly synthesized alphagamma(2) and alphabetagamma(2) receptors. We demonstrate that the presence of untrimmed N-linked core glycans (Glc(3)Man(9)GlcNAc(2)) on the FcepsilonRI alpha-chain activates the ER quality control mechanism to retain this subunit in the ER, despite the presence of gamma-chains. At the same time, the untrimmed, ER-localized alpha-chain exhibits IgE-binding activity, suggesting that FcepsilonRI alpha-chain folding occurs before constitutive glucose trimming. In additional experiments, we demonstrate that cell surface expression of an alpha-chain C-terminal truncation mutant is also dependent on glucose trimming, but not on gamma-chain coexpression. We suggest that glucosidase trimming of terminal glucose residues is a critical control step in the export of FcepsilonRIalpha from the ER. Finally, we show that the constitutive ER FcepsilonRI alpha-chain, expressed in the absence of the other FcepsilonRI subunits, associates with the ER lectin-like chaperone calnexin, but not the structurally similar ER chaperone calreticulin, presumably through interaction with monoglucosylated alpha-chain ER glycoforms.     

Allelic exclusion of the TCR alpha-chain is an active process requiring TCR-mediated signaling and c-Cbl

Phenotypic allelic exclusion at the TCRalpha locus is developmentally regulated in thymocytes. Many immature thymocytes express two cell surface alpha-chain species. Following positive selection, the vast majority of mature thymocytes and peripheral T cells display a single cell surface alpha-chain. A posttranslational mechanism occurring at the same time as positive selection and TCR up-regulation leads to this phenotypic allelic exclusion. Different models have been proposed to explain the posttranslational regulation of the alpha-chain allelic exclusion. In this study, we report that allelic exclusion is not regulated by competition between distinct alpha-chains for a single beta-chain, as proposed by the dueling alpha-chain model, nor by limiting CD3 zeta-chain in mature TCR(high) thymocytes. Our data instead favor the selective retention model where the positive selection signal through the TCR leads to phenotypic allelic exclusion by specifically maintaining cell surface expression of the selected alpha-chain while the nonselected alpha-chain is internalized. The use of inhibitors specific for Lck and/or other Src kinases indicates a role for these protein tyrosine kinases in the signaling events leading to the down-regulation of the nonselectable alpha-chain. Loss of the ubiquitin ligase/TCR signaling adapter molecule c-Cbl, which is important in TCR down-modulation and is a negative regulator of T cell signaling, leads to increased dual alpha-chain expression on the cell surface of double-positive thymocytes. Thus, not only is there an important role for TCR signaling in causing alpha-chain allelic exclusion, but differential ubiquitination by c-Cbl may be an important factor in causing only the nonselected alpha-chain to be down-modulated.     

Subunit structured of pig small-intestinal brush-border aminopeptidase N.

Aminopeptidase N (EC 3.4.11.2), when isolated from pig intestine in either the proteinase- or detergent-released form, frequently appears to contain three polypeptide chains, here termed alpha, beta and gamma. We have established by an immunological technique that the beta- and gamma-polypeptides are derived from the alpha-chain and that the intact enzyme is a dimer, alpha 2. Each alpha-chain of the detergent form was shown to contain a hydrophobic anchor peptide about 35 amino acid residues in length, which included the N-terminal sequences. A peptide bond in the alpha-chain was very sensitive to proteolysis. Its cleavage generated the commonly observed forms: alpha beta gamma and beta 2 gamma 2. The gamma-fragment, which lacked the anchor peptide, was derived from the C-terminal part of the alpha-chain.     

Identification of pathogenic T cells in patients with beryllium-induced lung disease.

Chronic beryllium disease (CBD) is caused by beryllium exposure and is characterized by granulomatous inflammation with accumulation of CD4+ T cells in the lung. We analyzed TCR beta-chain and alpha-chain genes expressed by these CD4+ T cells. In the lungs of individual patients, as well as among four of five CBD patients studied, different oligoclonal expansions within the Vbeta3 subset were found to express homologous or even identical CDR3 amino acid sequences. These related expansions were specific for CBD patients, were compartmentalized to lung, and persisted at high frequency in patients with active disease. Limiting dilution cloning and analysis of coexpressed TCR alpha-chain genes confirmed that these TCRs were selectively expanded by a common Ag involving beryllium. Overall, homologous TCR beta- and alpha-chains showed identical V regions and invariant charged residues within the CDR3 but considerable variability in TCRJ usage. Remarkably, CBD patients expressing nearly identical TCRs did not share common HLA-DRB1 or DQ alleles. These results implicate particular CD4+ cells in the pathogenesis of CBD and provide insight into how beryllium is recognized in human disease.     

Identification and cellular distribution of distinct proteins forming human GM-CSF receptor.

Two proteins forming the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF)1 were identified and characterized. One with apparent Mr of about 80,000 was defined as alpha-chain and has Kd of 0.7-2.8 nM. The other binding molecule with apparent Mr of about 135,000 was defined as beta-chain and is related to the high-affinity binding with Kd of 10-40 pM. The binding kinetic studies confirmed that the 125I-GM-CSF associated slower to and dissociated more rapidly from the alpha-chain than the beta-chain. The alpha-chain is expressed not only on hemopoietic cells but also on full-term placental tissues, choriocarcinoma cells, and other solid tumor cells. In contrast, the distribution of the beta-chain is restricted on hemopoietic cells. The alpha-chain probably corresponds to the low-affinity GM-CSF receptor whose cDNA has been cloned and sequenced.     

A deletion involving Alu sequences in the beta-hexosaminidase alpha-chain gene of French Canadians with Tay-Sachs disease

French Canadians living in eastern Quebec are carriers of a severe type of Tay-Sachs disease, known as the classic form, 10 times more often than the general population. The alpha-chain of beta-hexosaminidase A, a lysosomal enzyme composed of two chains (alpha, beta), bears the mutation in this inherited disorder. We previously reported that the 5' end of the alpha-chain gene was deleted in two such patients (Myerowitz, R., and Hogikyan, N.D. (1986) Science, 232, 1646-1648). The present study reports the size, precise location, and environment of the deletion. A clone encompassing the deletion was isolated from a genomic library constructed in lambda EMBL3 with DNA from a patient's fibroblasts. Comparison of the restriction maps of the clone with that of the normal gene (Proia, R.L., and Soravia, E. (1987) J. Biol. Chem. 262, 5677-5681) showed that the deletion was 7.6 kilobases long and included part of intron 1, all of exon 1 and extended 2000 base pairs upstream past the putative promotor region of the alpha-chain gene. These data are consistent with the inability to detect mRNA and immunoprecipitable alpha-chain protein in this mutant. Sequence analysis of the deletion junction in the mutant and corresponding regions of the normal gene demonstrated the presence of similarly oriented Alu sequences at the 5' and 3' deletion boundaries. The data are in accord with the possibility that the deletion may have arisen during homologous recombination from unequal crossing over between Alu sequences.