Imiquimod simultaneously induces autophagy and apoptosis in human basal cell carcinoma cells.

BACKGROUND: Imiquimod shows antitumour activity through the stimulation of cell-mediated immunity in vivo. Recent studies have shown that imiquimod promotes apoptosis in melanoma cells and induces autophagy in macrophage cell lines. OBJECTIVES: To evaluate the imiquimod-induced apoptosis, autophagy and their relationship in a basal cell carcinoma (BCC) cell line. METHODS: Cell viability was determined by XTT test. Apoptosis was evaluated by DNA content assay, annexin V/propidium iodide staining assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assay. Autophagy was determined by LC3 immunoblotting, EGFP-LC3 puncta formation and quantification of acidic vesicular organelles with acridine orange staining. The temporal and spatial differences of imiquimod-induced apoptosis and autophagy were examined by immunoblotting and simultaneously monitored by staining the EGFP-LC3 transfected cells with caspase 3 fluorogenic substrate. We inhibited the apoptosis and autophagy by pancaspase inhibitor and siRNA for Beclin 1 or Atg5, respectively, to evaluate the interplay between imiquimod-induced apoptosis and autophagy. RESULTS: We found that imiquimod induces autophagy and apoptosis in BCC cells in a time- and dose-dependent manner. Imiquimod not only induced EGFP-LC3 puncta formation for autophagy, but also simultaneously activated an apoptotic caspase cascade in the same cells. Both apoptosis and autophagy induced by imiquimod cooperate to cause BCC cell death. However, inhibition of imiquimod-induced apoptosis increased the strength of autophagy, and inhibition of imiquimod-induced autophagy further promoted cell apoptosis. CONCLUSIONS: This study not only demonstrates that imiquimod can directly induce autophagy and apoptosis in BCC cells, but also shows the cooperation and coordination between these two processes to induce cell death.

In islet-specific glucose-6-phosphatase-related protein, the beta cell antigenic sequence that is targeted in diabetes is not responsible for the loss of phosphohydrolase activity.

AIMS/HYPOTHESIS: There are three members of the glucose-6-phosphatase (G6Pase) family: (1) the liver/kidney/intestine G6Pase-alpha (encoded by G6PC), which is a key enzyme in glucose homeostasis; (2) the ubiquitous G6Pase-beta (encoded by G6PC3); and (3) the islet-specific G6Pase-related protein (IGRP, encoded by /G6PC2). While G6Pase-alpha and G6Pase-beta are functional glucose-6-phosphate hydrolases, IGRP possesses almost no hydrolase activity. This was unexpected since G6Pase-alpha is more closely related to IGRP than G6Pase-beta. Recently, amino acids 206-214 in IGRP were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes, suggesting that this peptide confers functional specificity to IGRP. We therefore investigated the molecular events that inactivate IGRP activity and the effects of the beta cell antigen sequence on the stability and enzymatic activity of G6Pase-alpha. METHODS: Studies were performed using site-directed mutagenesis and transient expression assays. Protein stability was evaluated by Western blotting, proteasome inhibitor studies and in vitro transcription-translation. RESULTS: We showed that the residues responsible for G6Pase activity are more extensive than previously recognised. Introducing the IGRP antigenic motif into G6Pase-alpha does not completely destroy activity, although it does destabilise the protein. The low hydrolytic activity in IGRP is due to the combination of multiple independent mutations. CONCLUSIONS/INTERPRETATION: The loss of catalytic activity in IGRP arises from the sum of many sequence differences. G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, which prevents targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells could dictate susceptibilities to diabetes.

Receptor binding kinetics of human IL-3 variants with altered proliferative activity.

The binding kinetics of native IL-3 and a set of truncated IL-3 variants to the alpha subunit of the IL-3 receptor (IL-3Ralpha) were studied using surface plasmon resonance. These variants, with amino acid substitutions at residues, 22, 42, 43, 45, 46, 113, or 116, have previously been identified to have altered capacity to stimulate cell proliferation compared to native IL-3(1-133). In this study, variants E43N and F113Y exhibited >100-fold slower association rates than IL-3(15-125) consistent with residues 43 and 113 being essential for the binding of IL-3 to the IL-3Ralpha. Variants G42A, G42D, Q45V, D46S, K116V, and K116W exhibited increased association rates (up to 15-fold relative to IL-3(15-125)) and decreased dissociation rates (up to 7-fold). The results demonstrate that both the association and dissociation rates for the binding of IL-3 to the IL-3Ralpha are altered by truncation and by amino acid substitution at individual sites. Intracellular signaling studies using K116W and E43N demonstrate that differences in the IL-3alpha binding characteristics are reflected in magnitude and kinetics of STAT5 phosphorylation.

Modulation of the binding affinity of myelopoietins for the interleukin-3 receptor by the granulocyte colony-stimulating factor receptor agonist.

Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.

Enhancement of the immunity to foot-and-mouth disease virus by DNA priming and protein boosting immunization.

Subunit vaccination is effective in eliciting humoral responses to a variety of viral antigens, however, it has not generated persistent protective immunity to foot-and-mouth disease virus (FMDV). In this study, we observed that priming mice with a DNA plasmid encoding VP1 of the FMDV O/Taiwan/97 capsid protein followed by boosting with a VP1 peptide conjugate (P29-KLH) resulted in production of not only high titers of antibodies but also antibodies with FMDV neutralizing activities. Moreover, the mice immunized in this manner cleared the virus from their sera in FMDV challenge experiments. Mice subjected to DNA plasmid priming and P29-KLH protein boosting had relatively higher ratio of IgG2a/IgG1 than those primed and boosted with P29-KLH conjugate. Addition of an oligodeoxynucleotide (ODN) containing immunostimulatory cytosine-phosphate-guanosine (CpG) motifs to P29-KLH conjugate also induced a higher ratio of IgG2a/IgG1 and significantly higher titer of neutralizing antibodies. These results indicate that treating animals with DNA plasmids priming and FMDV antigen(s) boosting may elicit immunity to FMD and this immune response may be augmented by CpG ODN.

Bivalent binding and signaling characteristics of Leridistim, a novel chimeric dual agonist of interleukin-3 and granulocyte colony-stimulating factor receptors.

Leridistim is a member of a novel family of engineered chimeric cytokines, myelopoietins, that contain agonists of both interleukin-3 (IL-3) receptors (IL-3R) and granulocyte colony-stimulating factor (G-CSF) receptors (G-CSFR). To more clearly understand Leridistim's function at the molecular level, binding to both IL-3R and G-CSFR and subsequent signaling characteristics have been delineated. The affinity of Leridistim for the human G-CSFR was found to be comparable to that of native G-CSF (IC(50) = 0.96 nM and 1.0 nM, respectively). Both Leridistim and G-CSF induced receptor tyrosine phosphorylation to a similar maximal level. Compared with native recombinant human IL-3 (rhIL-3), Leridistim was found to possess higher affinity for the IL-3R alpha chain (IL-3Ralpha) (IC(50) = 85 nM and 162 nM, respectively). However, the increase in Leridistim binding affinity to the functional, high-affinity heterodimeric IL-3Ralphabeta(c) receptor is lower than that observed with rhIL-3 (85 nM and 14 nM vs 162 nM and 3.5 nM, respectively). Leridistim induced tyrosine phosphorylation of beta(c) to a level comparable to native IL-3, and the level of JAK2 tyrosine phosphorylation in cells expressing both IL-3R and G-CSFR was comparable to that observed with IL-3 or G-CSF alone. The ability of Leridistim to interact with IL-3R and G-CSFR simultaneously was demonstrated using surface plasmon resonance analysis. These studies were extended to demonstrate that Leridistim exhibited a higher affinity for the IL-3R on cells that express both the IL-3Ralphabeta(c) and the G-CSFR (IC(50) = 2 nM) compared with cells that contain the IL-3Ralphabeta(c) alone (IC(50) = 14 nM). Leridistim binds to both IL-3R and G-CSFR simultaneously and has been shown to activate both receptors. The bivalent avidity may explain the unique biologic effects and unexpected potency of Leridistim in hematopoietic cells compared with rhIL-3 or G-CSF alone or in combination.

Stereospecific inhibition of CETP by chiral N,N-disubstituted trifluoro-3-amino-2-propanols.

Chiral N,N-disubstituted trifluoro-3-amino-2-propanols represent a recently discovered class of compounds that inhibit the neutral lipid transfer activity of cholesteryl ester transfer protein (CETP). These compounds all contain a single chiral center that is essential for inhibitory activity. (R,S)SC-744, which is composed of a mixture of the two enantiomers, inhibits CETP-mediated transfer of [(3)H]cholesteryl ester ([(3)H]CE) from HDL donor particles to LDL acceptor particles with an IC(50) = 200 nM when assayed using a reconstituted system in buffer and with an IC(50) = 6 microM when assayed in plasma. Upon isolation of the enantiomers, it was found that the (R,+) enantiomer, SC-795, was about 10-fold more potent than the mixture, and that the (S,-) enantiomer, SC-794, did not have significant inhibitory activity (IC(50) > 0.8 microM). All of the activity of the (S,-)SC-794 enantiomer could be accounted for by contamination of this sample with a residual 2% of the highly potent (R,+) enantiomer, SC-795. The IC(50) of (R,+)SC-795, 20 nM, approached the concentration of CETP (8 nM) in the buffer assay. These chiral N,N-disubstituted trifluoro-3-amino-2-propanols were found to associate with both LDL and HDL, but did not disrupt overall lipoprotein structure. They did not affect the on or off rates of CETP binding to HDL disk particles. Inhibition was highly specific since the activities of phospholipid transfer protein and lecithin cholesterol acyl transferase were not affected. Competition experiments showed that the more potent enantiomer (R)SC-795 prevented cholesteryl ester binding to CETP, and direct binding experiments demonstrated that this inhibitor bound to CETP with high affinity and specificity. It is estimated, based on the relative concentrations of inhibitor and lipid in the transfer assay, that (R)SC-795 binds approximately 5000-fold more efficiently to CETP than the natural ligand, cholesteryl ester. We conclude that these chiral N,N-disubstituted trifluoro-3-amino-2-propanol compounds do not affect lipoprotein structure or CETP-lipoprotein recognition, but inhibit lipid transfer by binding to CETP reversibly and stereospecifically at a site that competes with neutral lipid binding.

Electrospray/tandem mass spectrometry for quantitative analysis of lipid remodeling in essential fatty acid deficient mice.

A method utilizing electrospray ionization coupled with tandem mass spectrometry was developed as a facile and rapid method to identify and quantify lipid remodeling in vivo. Electrospray/tandem mass spectrometric analyses were performed on lipids isolated from liver tissue and resident peritoneal cells from essential fatty acid sufficient and deficient mice. Essential fatty acid deficiency was chosen as the paradigm to evaluate the methodology because it epitomizes the most extreme dietary means of altering fatty acid composition of virtually all cellular lipid species. Qualitative and quantitative changes were measured in the phospholipid and cholesterol ester species directly in the chloroform/methanol lipid extract without any prior chromatographic separation. Lipid remodeling in liver and peritoneal cells from essential fatty acid deficient mice was qualitatively similar in cholesterol ester, phosphatidylcholine, and phosphatidylethanolamine. The monoenoic fatty acids palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9) were increased markedly, whereas all n-6 and n-3 polyunsaturated fatty acids were nearly depleted in phospholipid and cholesterol ester species. The n-9 polyunsaturated fatty acid surrogate, Mead acid (20:3 n-9), substituted for arachidonic acid (20:4 n-6) and docosahexaenoic acid (22:6 n-3) in phospholipid, but not in cholesterol ester, species. Another notable difference was that adrenic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-6), both metabolites of arachidonic acid, accumulated in phospholipid and cholesterol ester species of peritoneal cells, but not in liver cells, of essential fatty acid sufficient mice. The overall body of data presented illustrates the implementation of electrospray/tandem mass spectrometry as a method for facile and direct quantification of changes in lipid species during lipid metabolic studies.

Surface plasmon resonance biosensor studies of human wild-type and mutant lecithin cholesterol acyltransferase interactions with lipoproteins.

Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.

Circular permutation of granulocyte colony-stimulating factor.

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.

Circular permutation of the granulocyte colony-stimulating factor receptor agonist domain of myelopoietin.

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.

Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.

Structural and functional properties of two mutants of lecithin-cholesterol acyltransferase (T123I and N228K).

Two naturally occurring mutants of human lecithin-cholesterol acyltransferase (LCAT), T123I and N228K, were expressed in COS-1 and Chinese hamster ovary cells, overproduced, and purified to homogeneity in order to study the structural and functional defects that lead to the LCAT deficiency phenotypes of these mutations. The mutants were expressed and secreted by transfected cells normally and had molecular weights and levels of glycosylation similar to wild type LCAT. The purified proteins (>98% purity) had almost indistinguishable structures and stabilities as determined by CD and fluorescence spectroscopy. Enzymatic activities and kinetic analysis of the pure enzyme forms showed that wild type LCAT and both mutants were reactive with the water-soluble substrate, p-nitrophenyl butyrate, indicating the presence of an intact core active site and catalytic triad. Both the T123I and N228K mutants had markedly depressed reactivity with reconstituted HDL (rHDL), but T123I retained activity with low density lipoprotein. To determine whether defective binding to rHDL was responsible for the low activity of both mutants with rHDL, the equilibrium binding constants were measured directly with isothermal titration calorimetry and surface plasmon resonance (SPR) methods. The results indicated that the affinities of the mutants for rHDL were only about 2-fold lower than the affinity of wild type LCAT (Kd = 2.3 x 10(-7) M). Together, the activity and equilibrium binding results suggest that the T123I mutant is defective in activation by apolipoprotein A-I, and the N228K mutant has impaired binding of lipid substrate to the active site. In addition, the kinetic binding rate constants determined by the SPR method indicate that normal LCAT dissociates from rHDL, on average, after one catalytic cycle.

Frequent mutation in Chinese patients with infantile type of GSD II in Taiwan: evidence for a founder effect.

Glycogen storage disease type II (GSD II, Pompe's disease), an autosomal recessive inherited disease, is caused by the deficiency of acid alpha-D-glucosidase, which results in the impaired glycogen degradation in lysosome and causes excess glycogen accumulation in lysosome. In Taiwan, the infantile form of GSD II is the most common type of glycogen storage diseases. The frequency of C1935A mutant allele is 0.8 in these Chinese patients. In this study, we analyzed four single point polymorphic markers (324, 1203, 2065, 2338) by ACRS-based RFLP We observed that the alleles possessing the C1935A mutation in 19 of 25 Chinese patients who were heterozygous or homozygous have conserved polymorphic markers, and all of C1935A mutant alleles in these patients are linked to a specific haplotype. The allele frequency of this specific haplotype in 19 Chinese patients and in 42 normal individuals is 0.95 and 0.17, respectively (P<0.005, chi2 = 66.018). This result suggests that the C1935A mutation in Chinese patients with infantile form of GSD II is due to the founder effect.

Molecular study on the infantile form of Pompe disease in Chinese in Taiwan.

Glycogen-storage disease type II, Pompe disease, is caused by the deficiency of acid alpha-D-glucosidase in lysosome. Previously we found that acid alpha-D-glucosidase did exist in the skin fibroblasts and there was also no difference of mRNA in quantity and size of Chinese infantile type Pompe disease patients in Taiwan. However, functional assay of the acid alpha-D-glucosidase of these patients showed its enzyme function to be defective. In the present study, first we identified a substitution site in four Chinese infantile patients with Pompe disease which is a cytidine to adenosine (C1935-->A) transversion at 5' end of exon 14 causing substitution of glutamic acid for aspartic acid at position 645 of the acid alpha-D-glucosidase. This substitution was introduced in wild-type cDNA and expressed in COS-1 cells. The Asp-645-->Glu substitution resulted in significant reduction of acid alpha-D-glucosidase activity. Second, according to the screening data in 25 Chinese Pompe disease patients using digestion of RT-PCR amplified specific fragment with Aat II, the restriction fragment length analysis showed that patients presented the 861 bp band and the normal individuals presented the 728 bp and 133 bp polymorphic bands. We found that the frequency of mutant allele is 0.8 in infantile patients with Chinese Pompe disease and 0 in normal individuals. These results therefore indicate that Asp-645-->Glu mutation results in infantile form of Pompe disease as the major cause in Chinese patients in Taiwan.

Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2).

CTLA-4 expressed on activated T cells binds to CD80 (B7-1) and CD86 (B7-2) molecules present on APC with high avidity and appears to deliver a negative regulatory signal to the T cell. We have investigated the kinetics of CTLA-4 binding to CD80 and CD86, together with the effects of selected CTLA-4 mutations on binding activity. The dissociation constants (Kd) for binding of CTLA-4-Ig to CD80 and CD86 transfectants were 8.1 and 6.7 nM, respectively. Surface plasmon resonance was used to determine kinetic parameters of CTLA-4-Ig binding to CD80-Ig and CD86-Ig fusion proteins and revealed enhanced association (ka) and dissociation (kd) rate constants for CD86-Ig compared with CD80-Ig. Furthermore, CD80-Ig and CD86-Ig fusion molecules demonstrated variable abilities to cross-compete for binding to several modified forms of CTLA-4-Ig. Differential binding of CD80 and CD86 to CTLA-4 was further revealed by analysis of 10 discrete CTLA-4 mutants. Five single amino acid substitutions within the CTLA-4 MYPPPY domain exerted modest effects on CD80 binding, but each of these substitutions completely abrogated CD86 binding. In addition, substitutions just N-terminal of the MYPPPY region, and within the CDR1-like region of CTLA-4, eliminated both CD80 and CD86 binding. Hence, CD80 and CD86 bind with different association/dissociation kinetics to similar, but distinct, sites on CTLA-4.

Identification of a small deletion in one allele of patients with infantile form of glycogen storage disease type II.

Glycogen storage disease type II (GSD II, Pompe's disease) is an autosomal recessive inherited disease caused by the deficiency of acid alpha-D-glucosidase. In this paper we report two unrelated Chinese patients with infantile form of GSD II who had compound heterozygotes containing a small deletion in one of the acid alpha-D-glucosidase alleles. In both of these compound heterozygotes, one allele contains the C1935A transversion which is the most common mutation in Chinese patients and the other allele contains the newly identified 4 nt deletion of coding sequence (deletion nt 1411-1414). This small deletion causes a reading frameshift and translational premature termination signal in exon 9.

Identification of a de novo point mutation resulting in infantile form of Pompe's disease.

One patient in a nonconsanguineous Taiwanese family with infantile glycogen storage disease type II (Pompe's) disease was found to have a de novo mutation of G1933 to C transition [corrected] in exon 14 of the human lysosomal alpha-D-glucosidase gene. Patient was homozygous and both parents were heterozygous for the mutant allele. The mutation caused an Asp to His substitution at amino acid position 645. The mutation was introduced in wild type lysosomal alpha-D-glucosidase cDNA and the mutant construct was expressed in vivo. The Glu to His substitution was proven to cause significant loss of enzyme activity. In homozygous form it leads to the severe infantile phenotype of Pompe's disease.