Age-dependent variation of genotypes in MHC II transactivator gene (CIITA) in controls and association to type 1 diabetes.

The major histocompatibility complex class II transactivator (CIITA) gene (16p13) has been reported to associate with susceptibility to multiple sclerosis, rheumatoid arthritis and myocardial infarction, recently also to celiac disease at genome-wide level. However, attempts to replicate association have been inconclusive. Previously, we have observed linkage to the CIITA region in Scandinavian type 1 diabetes (T1D) families. Here we analyze five Swedish T1D cohorts and a combined control material from previous studies of CIITA. We investigate how the genotype distribution within the CIITA gene varies depending on age, and the association to T1D. Unexpectedly, we find a significant difference in the genotype distribution for markers in CIITA (rs11074932, P=4 × 10(-5) and rs3087456, P=0.05) with respect to age, in the collected control material. This observation is replicated in an independent cohort material of about 2000 individuals (P=0.006, P=0.007). We also detect association to T1D for both markers, rs11074932 (P=0.004) and rs3087456 (P=0.001), after adjusting for age at sampling. The association remains independent of the adjacent T1D risk gene CLEC16A. Our results indicate an age-dependent variation in CIITA allele frequencies, a finding of relevance for the contrasting outcomes of previously published association studies.

Identification and characterization of a novel isoform of the vesicular gamma-aminobutyric acid transporter with glucose-regulated expression in rat islets.

Pancreatic islets are unique outside the nervous system in that they contain high levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), synthesized by the enzyme glutamic acid decarboxylase (GAD). Since the role that GABA plays in the islet and the mechanisms whereby the two major GAD isoforms (GAD65 and GAD67) function as diabetes-associated autoantigens are unknown, continued characterization of the islet GAD-GABA system is important. We previously demonstrated that the GABA and glycine transporter vesicular inhibitory amino acid transporter (VIAAT also known as VGAT) is present in rat islets. Here we identify a novel 52 kDa variant of VIAAT in rat islets: VIAAT-52 (V52). V52 is an amino-terminally truncated form of VIAAT (V57) that likely results from utilization of a downstream start site of translation. V57 and V52 display different patterns of post-translational modification and cellular expression. Our results have indicated that islet content of V52, but not V57, is responsive to changes in glucose concentration and other extracellular conditions. VIAAT is expressed in the islet alpha cells, but there have been conflicting findings regarding the presence of VIAAT in the beta cells. Here we have also provided additional evidence for the presence of VIAAT in islet beta cells and show that the beta cell line INS-1 expresses V57. V52 may be better adapted than V57 to the unique rat alpha cell GAD-GABA system, which lacks GAD65 and in which VIAAT traffics to secretory granules rather than just to synaptic microvesicles.

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.

Cleavage of the transferrin receptor is influenced by the composition of the O-linked carbohydrate at position 104.

A soluble form of the human transferrin receptor (TfR) resulting from proteolytic cleavage at Arg 100 has been measured in human blood. In tissue culture cells elimination of the O-linked carbohydrate at Thr 104, four amino acids from the cleavage site, results in enhanced cleavage of the TfR (Rutledge et al., 1994, Blood, 83:580-586). In the present set of studies, the influence of amino acid substitution and the composition of the oligosaccharide at amino acid 104 on the cleavage of the TfR was examined. Site-directed mutagenesis was used to generate six different amino acids at position 104 which varied in size and charge. Measurement of the soluble TfR in the conditioned medium of the transfected cells of each mutant TfR showed that the large and charged side chains inhibited TfR cleavage the most. Otherwise the properties of the mutant TfRs were indistinguishable from the wild-type TfR in that the affinity of transferrin for these receptors, the extent of disulfide bond formation of the TfRs, and the proportion of TfRs at the cell surface were similar to that of the wild-type TfR. Removal of the sialic acid component of the carbohydrate from wild-type TfR by treatment of live cells with neuraminidase enhances TfR cleavage. Expression of wild-type TfR in CHO IdlD cells (a glycosylation defective cell line) also shows enhanced cleavage under conditions that produce truncated or no O-linked carbohydrates. Treatment of IdlD cells with neuraminidase reveals that the sialic acid of the O-linked carbohydrate protects against TfR cleavage, whereas the core sugars Gal-NAc and Gal do not protect as much. These results show that the terminal charged sialic acid residues are important for protection from proteolytic cleavage and suggest that cleavage could be regulated in the cell by removal of all or part of the carbohydrate.

Generation of the soluble transferrin receptor requires cycling through an endosomal compartment.

The transmembrane protein, transferrin receptor (TfR), is found in a soluble form in human serum and in the medium of cell lines grown in tissue culture. The soluble form is generated by proteolytic cleavage between Arg-100 and Leu-101. We used two mutant human TfRs expressed in Chinese hamster ovary (CHO) cells lacking endogenous transferrin receptor to characterize the protease that cleaves the TfR and determine its location in the cell. The T104D mutant TfR lacks the O-linked carbohydrate at position 104, and is more susceptible to proteolytic cleavage at Arg-100 than the wildtype human TfR in these cells. We find that the protease is not a component of the serum in the growth medium, and it is not secreted by the cells. Cleavage does not occur during biosynthesis of the TfR, and occurs after the TfR has reached the cell surface. Expression of the T104D TfR in a temperature-sensitive acidification defective CHO cell line, G.7.1, shows that cleavage of the TfR is not dependent on acidification of endosomes. The C20A23 TfR is an endocytosis deficient mutant lacking an internalization signal. This mutant TfR, which is mainly localized to the cell surface, is cleaved less efficiently than the wild-type TfR, indicating that the protease is localized to an intracellular compartment.

Turnover of the transferrin receptor is not influenced by removing most of the extracellular domain.

We treated intact cells with trypsin to remove most of the external domain of the transferrin receptor and investigated what effect the absence of the external domain had on the turnover of the fragment that remained associated with the cells. To detect the cell-associated tryptic fragment, which contains a small amount of the external domain, the transmembrane domain, and the cytoplasmic domain, we prepared an anti-peptide antibody against a segment of the cytoplasmic domain. This antibody specifically immunoprecipitated the intact transferrin receptor as well as a 21-kDa peptide from trypsin-treated HeLa cells. Several lines of evidence indicated that the 21-kDa peptide was the cell-associated tryptic fragment of the transferrin receptor. The fragment was only present in trypsin-treated cells; the fragment migrated as a dimer in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as it should if it were derived from the transferrin receptor; a goat antibody prepared to the purified human transferrin receptor also precipitated the 21-kDa peptide from trypsinized cells. In addition, treating the tryptic fragment with neuraminidase increased the electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting the fragment contained O-linked carbohydrate. When cells were trypsinized and then incubated at 37 degrees C, the half-life of the tryptic fragment (15 +/- 4 h) was not significantly different than the half-life of the intact receptor (19 +/- 6 h). This indicates that removing 95% of the external domain of the transferrin receptor has little effect on processes operating in the turnover of the receptor.

Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2.

Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.

The transferrin receptor cytoplasmic domain determines its rate of transport through the biosynthetic pathway and its susceptibility to cleavage early in the pathway.

The soluble human transferrin receptor (TfR) found in blood is the result of a proteolytic cleavage occurring in the ectodomain of the receptor close to the transmembrane domain at Arg-100. We have discovered another cleavage site between Gly-91 and Val-92 even closer to the transmembrane domain. Cleavage at Gly-91 differs markedly from the normal cleavage site. It occurs when the entire cytoplasmic portion or the proximal 31 amino acids of the transmembrane domain are deleted. A soluble disulfide-bonded dimer of the TfR is released into the medium in contrast to the cleavage at Arg-100 where a dimer lacking intersubunit disulfide bonds is released. Whereas the cleavage at Arg-100 is generated by cycling through the endosomal system, pulse-chase experiments indicate that cleavage at Gly-91 occurs predominantly during the biosynthesis of the receptor. Pulse-chase analysis of the biosynthesis of mutant TfRs that lack the membrane-proximal cytoplasmic domain show that they exit the endoglycosidase H-sensitive compartment at a slower rate than the wild type TfR. These results suggest that the cytoplasmic domain influences the trafficking of the TfR either by influencing the folding of the ectodomain or by providing a positive signal for its transport through the biosynthetic pathway.

Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor.

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron-transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes.

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.

Impaired secretion and fluid-phase endocytosis in the End4 mutant of Chinese hamster ovary cells.

Mutant V.24.1 defines the End4 complementation group of temperature-sensitive Chinese hamster ovary cell mutants selected for resistance to protein toxins. We investigated the secretory pathway in the mutant cells and found: 1) The hemagglutinin of influenza virus failed to reach the plasma membrane and was retained in a form sensitive to endoglycosidase H at the restrictive temperature. 2) Transferrin receptors synthesized at the restrictive temperature remained sensitive to endoglycosidase H. 3) Secretion of total soluble protein into the medium was strongly reduced at high temperature. These data indicate that V.24.1 cells are defective in secretion at the restrictive temperature. To see what effect the lesion had on the endocytic pathway, we measured the accumulation and recycling of the fluid-phase marker horseradish peroxidase. Accumulation was inhibited by 50% while recycling was barely affected, suggesting that the rate of fluid-phase endocytosis was reduced. We previously showed that the clathrin-coated pit pathway of endocytosis was not affected in the mutant, indicated by a normal transferrin cycle (Colbaugh, P. A., Stookey, M., and Draper, R. K. (1989) J. Cell Biol. 108, 2211-2219). Thus, the secretory lesion correlates with reduced fluid-phase endocytosis without impairing the clathrin-dependent pathway of receptor-mediated endocytosis. We also investigated the delivery of endocytosed material to lysosomes and found that delivery was partially, but not completely, impaired in the mutant. This suggests that endocytosed material can enter lysosomes, although slowly, in the absence of a functional secretory pathway.