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Biomedical subjects

J Trowsdale

Publications and source records attributed to J Trowsdale.

At least 91 records · Page 5Linked to original sources
PubMed1997

Dynamics of proteasome distribution in living cells.

Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.

Antigen Presentation↗
PubMed1997

A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes.

Newly assembled major histocompatibility complex (MHC) class I molecules, together with the endoplasmic reticulum chaperone calreticulin, interact with the transporter associated with antigen processing (TAP) through a molecule called tapasin. The molecular cloning of tapasin revealed it to be a transmembrane glycoprotein encoded by an MHC-linked gene. It is a member of the immunoglobulin superfamily with a probable cytoplasmic endoplasmic reticulum retention signal. Up to four MHC class I-tapasin complexes were found to bind to each TAP molecule. Expression of tapasin in a negative mutant human cell line (220) restored class I-TAP association and normal class I cell surface expression. Tapasin expression also corrected the defective recognition of virus-infected 220 cells by class I-restricted cytotoxic T cells, establishing a critical functional role for tapasin in MHC class I-restricted antigen processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Physical mapping 220 kb centromeric of the human MHC and DNA sequence analysis of the 43-kb segment including the RING1, HKE6, and HKE4 genes.

A cosmid contig was constructed from a YAC clone with a 220-kb insert that spans the centromeric side of the human MHC class II region, corresponding to the mouse t complex. The gene order was identified to be HSET-HKE1.5-HKE2-HKE3-RING1-HKE6- HKE4 (RING5). The genomic sequence of a 42,801-bp long region encoded by one cosmid clone in the RING1, HKE6, and HKE4 subregions was determined by the shotgun method. The exon-intron organization of these three genes, RING1 (Ring finger protein), HKE6 (steroid dehydrogenase-like protein), and HKE4 (transmembrane protein with histidine-rich charge clusters), was determined. The previously reported RING2 gene was revealed to be identical to HKE6. Transcripts from HKE4 were detected in the placenta, lung, kidney, and pancreas. Those of HKE6 were found in the liver and pancreas. The 25-kb region proximal to the RING1 gene includes an extensive dense cluster of Alu repeats (about 1.2 Alu per kb), and no gene has been identified in this so far. The region is equivalent to part of the mouse t complex and could be of relevance to human development.

Amino Acid Sequence↗
PubMed1997

Cloning of NKG2-F, a new member of the NKG2 family of human natural killer cell receptor genes.

The NKG2 family of genes encodes at least four different type II transmembrane molecules (NKG2-A, NKG2-B, NKG2-C and NKG2-E) which contain a C-lectin domain. These proteins have been shown to be covalently associated with CD94, another C-type lectin member. The heterodimers are involved in natural killer cell-mediated recognition of different HLA-allotypes. Here we describe the cloning of a new NKG2-related gene, termed NKG2-F, localized 25 kb from NKG2-A as well as its relationship with the previously described NKG2-D cDNA. Despite the similarities with the other NKG2 genes, NKG2-F encodes a putative protein which does not contain any lectin domain. However, a conserved 24-amino acid sequence, present in all members of the NKG2 family, suggests that NKG2-F is also able to form heterodimers with CD94.

Amino Acid Sequence↗
PubMed1997

Two widely used anti-DR alpha monoclonal antibodies bind to an intracellular C-terminal epitope.

In this report we show that two widely-used monoclonal antibodies, TAL-1B5 and DA6.147, which react with the HLA-DR alpha chain on immunoblots, recognize the C-terminal intracellular tail of this HLA-DR subunit. We demonstrate that both MoAbs react with a synthetic peptide representing the intracellular C-terminal tail of the DR alpha chain and that mutant DR molecules lacking this part of the alpha chain lose reactivity with TAL-1B5 and DA6.147, both in Western blot analysis and in intracellular FACS staining.

Amino Acid Sequence↗
PubMed1997

Generation of novel human MHC class II mutant B-cell lines by integrating YAC DNA into a cell line homozygously deleted for the MHC class II region.

The human B lymphoblastoid cell line (LCL) 721.174 sustains a large homozygous deletion in the major histocompatibility complex (MHC) class II region that results in an absence of DQ and DR molecules as well as a deficiency in the assembly and transport of class I molecules to the cell surface. The deleted genes include the transporters associated with antigen processing TAP1 and TAP2, DMA and DMB which are involved in editing class II bound peptides, and four genes whose roles in antigen processing are unclear; the low mass polypeptide genes LMP2 and LMP7, and DNA and DOB. To study this region we have integrated into 721.174 two overlapping yeast artificial chromosome (YAC) clones which cover the interval LMP2-DRA inclusive. Three clones (11.2A1.1, 4D1D10.1 and 4D1D10.2), containing complete copies of the transfected YAC, produced varying levels of mRNA from the LMP, TAP, DQ and DR genes and corresponding levels of LMP and TAP protein. Class I cell surface expression was restored in 11.2A1.1 and 4D1D10.1, as was DR expression in both 4D1D10 transfectants. These studies demonstrate the feasibility of introducing large groups of functional genes back into human lymphoblastoid cells sustaining deletions, with full restoration of biological function. The procedure could be exploited in order to restore all but one gene covered by the deletion, effectively producing a single gene defect. This could be used to introduce copies of genes engineered to contain mutations and to study cis regulatory elements at some distance from the chosen loci.

Amino Acid Sequence↗
PubMed1997

Genomic organization of a human killer cell inhibitory receptor gene.

We have cloned a region of human chromosome 19q13.4 which contains multiple killer cell inhibitory receptor (KIR) loci. By random and directed sequence analysis of these KIR-specific clones, we deduced the genomic structure of KIR genes. A locus encoding a member of the NKAT-2 family of KIRs is presented here. The structure of the gene is reminiscent of loci of the Fc receptor gene family, and the two sets of genes may derive from a common ancestor. The KIR gene contains potentially nine exons. The first two exons encode the leader sequence, as in Fc receptor genes. The third exon encodes an untranslated pseudo exon specifying an immunoglobulin domain with an in-frame stop codon. Expressed cDNAs do not contain this exon. This finding is consistent with the hypothesis that certain KIR genes may have been derived from the duplication of a primordial three immunoglobulin domain structure with subsequent skipping of one exon to derive genes with two expressed immunoglobulin domains. Variation in numbers of immunoglobulin domains in different KIR genes is facilitated by conservation of splicing frame in respect to the codon triplet for each immunoglobulin domain.

Alternative Splicing↗
PubMed1997

Proteasome subunits, low-molecular-mass polypeptides 2 and 7 are hyperexpressed by target cells in autoimmune thyroid disease but not in insulin-dependent diabetes mellitus: implications for autoimmunity.

Autoimmune thyroid diseases (AITD) and insulin-dependent diabetes mellitus (IDDM) are two autoimmune syndromes of unknown etiology with common immune features. One is that the target cells, thyrocytes and pancreatic islet beta cells respectively, hyperexpress several proteins encoded in the HLA region: HLA class I, HLA class II and transporter associated with antigen processing (TAP-1): the clinical course and many aspects of the immunopathology are, however, quite different. Low-molecular-mass polypeptides 2 and 7 (LMP2 and LMP7) are proteasome subunits that increase the efficiency of endogenous antigen processing and are encoded in close vicinity to the TAP genes. We investigated whether LMP2 and LMP7 are hyperexpressed in thyrocytes and islet cells in AITD and IDDM. Thyroid tissue from Graves' disease patients (GD, n = 8) and Hashimoto thyroiditis (HT, n = 1) and pancreatic tissue from IDDM patients (n = 4) as well as control tissues were examined by the two-color indirect immunofluorescence technique. The results demonstrate that, in normal glands, thyrocytes and pancreatic islet cells express comparable moderate to low levels of LMP2 and LMP7. In AITD and IDDM, expression of LMP2/7 in the endocrine cells was disparate: while in AITD glands there was hyperexpression of LMP2 and 7 parallel to that of HLA class I and TAP-1, in the islet cells of recent onset diabetic pancreases (n = 2) the level of LMP2 and 7 expression was totally normal, including islets that were infiltrated by lymphocytes and hyperexpressed HLA class I and TAP-1. These observations suggest different mechanisms of endogenous peptides generation at the target cells in AITD from IDDM. Since this is a key step for the maintenance of peripheral tolerance, it may help to understand some of the different clinical features of the two autoimmune diseases.

Adolescent↗
PubMed1997

Homozygosity for the predominant Cys282Tyr mutation and absence of disease expression in hereditary haemochromatosis.

A candidate gene for hereditary haemochromatosis, HLA-H, has recently been presented. Two missense mutations in the HLA-H gene sequence are predicted to account for nearly 90% of all cases of the disease. The aim of this study was to correlate the presence of these missense mutations with the expressivity of the disease, as assessed by standard biochemical evaluation of serum iron parameters. Detection of the known mutations in haemochromatosis, Cys282Tyr and His63Asp, was undertaken in a large pedigree showing variable expression of the disease in successive generations. In three sibs with overt disease (one male, two female, aged 50 to 53 years), homozygosity for the predominant G to A transition (Cys282Tyr) in HLA-H was detected. However, homozygosity for this mutation was also detected in an asymptomatic male sib, aged 50, harbouring an identical genotype. The finding of an asymptomatic homozygous Cys282Tyr subject, haplo-identical to affected sibs, indicates that clinical expression of symptomatic disease is variable, even in middle aged Cys282Tyr homozygotes. This has profound implications for the future use of genetic screening for haemochromatosis.

Adult↗
PubMed1997

Complex duplications at 6p22.1, 6p11.2, 5q13, 5p15.1 and 5p13 revealed by fluorescent in situ hybridisation.

A complex pattern of fluorescent in situ hybridisation (FISH) has been detected using PAC clones from the short arm of chromosome 6, proximal to the haemochromatosis gene at 6p22.1. Cross-hybridisation to 6p22.1, 6p11.2, 5q13, 5p15.1 and 5p13 was consistently detected with several PAC clones covering a genomic region greater than 200 kb. These results indicate that large sections of genomic DNA are shared by these 5 disparate chromosomal segments, indicative of large scale duplication events. These results were in part accounted for by the identification of several expressed sequence tags (ESTs).

Chromosomes, Human, Pair 5↗
PubMed1997

Characterization of recombination in the HLA class II region.

Studies of linkage disequilibrium across the HLA class II region have been useful in predicting where recombination is most likely to occur. The strong associations between genes within the 85-kb region from DQB1 to DRB1 are consistent with low frequency of recombination in this segment of DNA. Conversely, a lack of association between alleles of TAP1 and TAP2 (approximately 15 kb) has been observed, suggesting that recombination occurs here with relatively high frequency. Much of the HLA class II region has now been sequenced, providing the tools to undertake detailed analysis of recombination. Twenty-seven families containing one or two recombinant chromosomes within the 500-kb interval between the DPB1 and DRB1 genes were used to determine patterns of recombination across this region. SSCP analysis and microsatellite typing yielded identification of 127 novel polymorphic markers distributed throughout the class II region, allowing refinement of the site of crossover in 30 class II recombinant chromosomes. The three regions where recombination was observed most frequently are as follows: the 45-kb interval between HLA-DNA and RING3 (11 cases), the 50-kb interval between DQB3 and DQB1 (6 cases), and an 8.8-kb segment of the TAP2 gene (3 cases). Six of the 10 remaining recombinants await further characterization, pending identification of additional informative markers, while four recombinants were localized to other intervals (outliers). Analysis of association between markers flanking HLA-DNA to RING3 (45 kb), as well as TAP1 to TAP2 (15 kb), by use of independent CEPH haplotypes indicated little or no linkage disequilibrium, supporting the familial recombination data. A notable sequence motif located within a region associated with increased rates of recombination consisted of a (TGGA)12 tandem repeat within the TAP2 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Human histocompatibility leukocyte antigen (HLA)-DM edits peptides presented by HLA-DR according to their ligand binding motifs.

Human histocompatibility leukocyte antigen (HLA)-DM is a facilitator of antigen presentation via major histocompatibility complex (MHC) class II molecules. In the absence of HLA-DM, MHC class II molecules do not present natural peptides, but tend to remain associated with class II-associated invariant chain peptides (CLIP). Recently, DM was shown to catalyze the release of CLIP from HLA-DR. We have investigated which peptides bound to HLA-DR are vulnerable to release upon encountering DM. By directed substitution of allele-specific anchor residues between CLIP and DR3-cognate peptides and the application of recombinant DM we show that DM catalyzes the release of those peptides bound to HLA-DR3 that do not have appropriate anchor residues and, hence, no optimal ligand binding motif. Thus, HLA-DM acts as a peptide editor, facilitating selection of peptides that stably bind to class II molecules for eventual presentation to the immune system from the pool of available peptides.

Amino Acid Sequence↗
PubMed1996

The TAP complex influences allorecognition of class II MHC molecules.

The influence of the TAP complex on T-cell allorecognition of MHC class II molecules was examined using human B-cell lines that have mutations in the TAP 1 or 2 genes. The TAP mutations led to the loss of allorecognition for two of 28 anti- HLA-DR T-cell clones. Restoration of TAP expression by transfection of a TAP 2 cDNA clone led to recovery of the alloresponse for both clones. These results could be explained in two ways. First, TAP dependence could reflect specificity for a peptide derived from an MHC class I molecule that is less efficiently generated by the endocytic pathway in the TAP-deficient stimulator cells owing to reduction in surface class I expression. The proliferative responses of these clones to the TAP-deficient stimulator cells was not restored by rescue of cell-surface expression of class I molecules by low temperature culture or by the addition of class I-binding peptides. These data therefore favor the alternative explanation that class II loading by some peptides is TAP dependent. Circumstances that lead to the amplification of this minority pathway of endogenous presentation by class II MHC molecules may have the potential to interrupt self-tolerance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Targeting signal and subcellular compartments involved in the intracellular trafficking of HLA-DMB.

Evidence suggests that peptide loading onto MHC class II molecules occurs in a specialized late endocytic compartment (MIIC) where HLA-DM predominantly resides and in which MHC class II transiently accumulates before transport to the cell surface. We examined the targeting signals and compartments involved in the intracellular trafficking of human HLA-DM by expressing hybrid molecules comprising the cytoplasmic domain of DMB and luminal and transmembrane domains of CD8 in HeLa cells. A tyrosine-based tetrapeptide motif present in the cytoplasmic domain of DMB targeted hybrid molecules to intracellular vesicles. Mutation of the tyrosine residue to alanine resulted in redistribution of hybrid molecules to the cell surface. Correct intracellular targeting of HLA-DM was crucial for normal function in B cells. Immunoelectron microscopy on ultrathin cryosections showed that CD8-DMB molecules accumulated in late endocytic compartments sharing characteristics with lysosomes, like MHC class II compartments in APCs. Thus far, the exit of DMB from the Golgi complex has not been elucidated. Interestingly, we found that although the mannose 6-phosphate receptor and CD8-DMB contain similar tyrosine signals, no co-localization was observed in the trans-Golgi network, suggesting that these proteins are differentially sorted at this site. Co-transfection of CD8-DMB, HLA-DR alpha, HLA-DR beta, and an invariant chain revealed that HLA-DR molecules accumulated together with CD8-DMB in these lysosomal compartments. The similarity of these lysosomal-like compartments in wild-type and transfected cells suggests that they are part of the normal endocytic pathway in non-APCs.

Amino Acid Sequence↗
PubMed1996

Evolutionary dynamics of non-coding sequences within the class II region of the human MHC.

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.

Amino Acid Sequence↗
PubMed1996

Divergent intron arrangement in the MB1/LMP7 proteasome gene pair.

We sequenced the human MB1 gene from a cosmid clone mapping to chromosome 14q11.2-12. The gene spans about 6 kilobases and contains three exons and two introns. There was no evidence of an alternative leader exon, which is a characteristic of the major histocompatibility complex (MHC)-encoded LMP7 gene, the closest relative of MB1, with which it shares 67% amino acid identity. Conceptual translation of the 5' end of the gene calls for a cleaved leader sequence of 59 amino acids, consistent with western blot data. None of the MB1 gene's three exons were coincident with any of the six exons in LMP7. In contrast, in the delta-encoding gene and its counterpart, the MHC-encoded LMP2 gene (59% amino acid identity), all six exons are arranged at equivalent positions in respect to the coding frame. The unique structure of MB1 implies a separate origin or different selection pressures acting at this particular locus. DNA repeat analysis provides information on the minimum time of separation of the MB1/LMP7 pair of genes.

Base Sequence↗