Heinz-Günter Thiele

Molecular characterization of mouse T-cell ecto-ADP-ribosyltransferase Rt6: Cloning of a second functional gene and identification of the Rt6 gene products

RT6 is an enzymatically active GPI-anchored membrane protein that was originally discovered in the rat as a peripheral T cell alloantigen. It has attracted interest as an activation antigen and because defective RT6-expression coincides with increased susceptibility for autoimmune type I diabetes in the BB rat. Southern blot analyses indicate that the rat carries a single copy RT6 gene whereas the mouse carries a duplication of the homologous locus. We had previously cloned and sequenced a RT6-homologous cDNA from BALB/c mouse spleen. We now report the cloning and characterization of a second RT6-homologue from BALB/c and 129/Sv mice. The two mouse Rt6 genes (designated Rt6-1 and Rt6-2) encode similar open reading frames that are disrupted by conserved introns. The nucleotide sequences of the Rt6-1 and Rt6-2 coding regions show 87% sequence identity, the deduced amino acid sequences 79% identity. The amino acid sequences reveal significant similarity to recently cloned ADP-ribosylating ectoenzymes from rabbit and human skeletal muscle as well as chicken bone marrow cells. RT-PCR analyses reveal that the two Rt6 genes are differentially expressed in distinct inbred mouse strains and that their transcripts are properly processed. Western blot analyses demonstrate that the respective gene products are released from cells by treatment with PI-PLC. The results further show that both mouse Rt6 genes are translated into GPI-anchored cell surface molecules and that Rt6 gene expression is restricted to peripheral lymphoid tissues.

Expression and comparative analysis of recombinant rat and mouse RT6 T cell mono(ADP-ribosyl)transferases in E. coli.

Recombinant RT6 proteins of rat and mouse were analyzed for NAD-metabolizing, i.e. mono(ADP-ribosyl)transferase, NAD-glycohydrolase (NADase) and ADP-ribosyl cyclase activities. The results reveal surprising intra- as well as inter-species differences in enzyme activities. While mouse Rt6 proteins were found to be strong arginine-specific transferases, but comparatively weak NADases, the opposite held true for rat RT6, for which transferase activity could only be detected in the form of arginine-specific auto-ADP-ribosylation, displayed by RT6.2 but not by RT6.1. NADase activity of rat RT6 was not accompanied by production of cyclic ADPR (cADPR). Rat RT6 gained potent arginine-specific transferase activity by exchange of a single amino acid for the corresponding residue of the mouse proteins.

The RT6 system of the rat: developmental, molecular and functional aspects

Summary: RT6 is a developmentally regulated cell‐surface membrane adenosine 5′‐diphosphate‐ribosyltransferase/nicotinamide adenine dinucleotide‐glycohydrolase inserted within the membrane by a glycophosphatidylinositol anchor. In the rat it is restricted to mature T lymphocytes and a subpopulation of natural killer cells. With respect to the data now available, three aspects concerning the function of RT6 are discussed: first, the meaning of the marked polymorphisms; second, its enzymatic activity; third, its possible role concerning T‐cell survival. The observation that the rat RT6 gene contains two transcription start sites suggests their different use by distinct subpopulations of T cells. The fact that the expression of RT6 is defective in lymphopenic diabetes prone (DP‐BB) rats, although the RT6 gene is structurally not grossly altered in these animals, makes this rat strain a promising model to study the biological meaning of RT6. While it mostly is believed that the RT6 expression defect of the DP‐BB rat is a consequence of the lymphopenia, the present paper discusses the possibility that the RT6 expression defect is causally involved in the lymphopenia, and that a normal expression of RT6 may protect the recent thymic emigrants from apoptosis.

Use of the EST Database Resource to Identify and Clone Novel Mono(ADP-Ribosyl)Transferase Gene Family Members

We searched the database of expressed sequence tags (dbEST) for relatives of the known human and murine mono(ADP-ribosyl)transferases (mADPRT), poly(ADP-ribosyl)polymerases (PARP), ADP-ribosyl cyclases, and ADP-ribosylarginine hydrolases (ARH). By May 31, 1996, all of the known enzymes except for RT6 were represented in dbEST by exact sequence matches from mouse and/or human tissues. Several ESTs show significant sequence similarity but not identity to known mADPRTs. We isolated, cloned, and sequenced the corresponding genes. Our results show that seven human ESTs stem from a novel gene, provisionally designated LART, which is specifically expressed in lymphatic tissues. Five human ESTs stem from a novel gene, here designated TART1, which is specifically expressed in testis. This gene is also represented by a single mouse EST. One other mouse EST stems from a distinct gene, here designated TART2, which is also expressed in testis. These genes have similar exon/intron structures. The predicted LART and TART1 gene products contain hydrophobic N- and C-terminal signal peptides characteristic for GPI-anchored surface proteins, TART2 lacks the GPI-anchor signal peptide. The predicted native proteins show 28-42% sequence identity to one another. They each contain four cysteine residues that probably form conserved disulfide bonds. They each also contain a conserved glutamic acid residue within the proposed active site motif LART and TART1 show interesting deviations from the surrounding consensus sequence.

Molecular polymorphism in the Rt6 genes of laboratory mice correlates with the allotypes of the H1 minor histocompatibility system

Minor H (histocompatibility) loci govern immune responses causing slow skin-graft rejection and graft versus host disease in major histocompatibility complex (MHC)-compatible animals. It is generally assumed that minor H antigens represent allelic variants of normal proteins and that peptides bearing the allelic amino acid substitutions are presented by MHC molecules in the context of which they may be recognized by H-allotype-specific T cells. 24 refs., 1 fig., 2 tabs.

A single-step purification procedure and partial amino acid sequence analysis of picomole amounts of the rat T cell alloantigen RT6.2

A single-step immunoaffinity purification procedure for the rat T cell marker RT6.2 is described which permits the isolation of microgram quantities of protein from the RT6.2+ T-T hybridoma EpD3. The amino terminus was sequenced directly from a polyvinylidene (PVDF) membrane blot prepared after SDS-PAGE. Further internal sequence data were obtained from peptides generated from purified RT6.2 digested with different endoproteases and separated by reverse-phase micro-HPLC. A computer search in data banks did not reveal any significant homology to other proteins.

Using secondary structure predictions and site-directed mutagenesis to identify and probe the role of potential active site motifs in the RT6 mono(ADP-ribosyl)transferases.

The RT6 T cell mono(ADP-ribosyl)transferases are expressed as GPI-anchored membrane proteins by mature T lymphocytes. We performed secondary structure prediction analyses of RT6 with a profile based neural network system based on multiple alignments of RT6 with other vertebrate mono(ADP-ribosyl)transferases (mADPRTs). The results reveal a linear order of predicted beta sheets/alpha helix in RT6 that are quite similar to those in the catalytic subunit of the four known crystal structures of mono-ADP-ribosylating bacterial toxins. Recognizable amino acid similarities occur throughout the region of predicted structural homology to the bacterial toxins. Three residues which have been shown to be important for catalysis in bacterial toxins (e.g. R9, S52 and E129 in pertussis toxin) occur in a similar context also in RT6 (R126, S147 and E189). We have mutated these residues in RT6 by site-directed mutagenesis. The RT6 mutants exhibit remarkably similar alterations in enzymatic phenotype as those reported for mutations of the proposed analagous residues in bacterial toxins. These results support the hypothesis that eu- and procaryotic mADPRTs share a common fold and have a common ancestry.

Molecular Cloning and Characterization of the T-CELL Mono(ADP-Ribosyl)Transferase RT6

The RT6 system (1) was first detected and serologically defined on peripheral T cells of different inbred strains of the Norway rat (2-6). It gained particular interest when it was reported that the diabetes-prone BioBreeding rat (DP-BB), which develops insulindependent diabetes mellitus (IDDM) between postnatal day 60 and 120 in a high frequency, does not appropriately express RT6 on the surfaces of its T cells (7). Subsequently RT6 -homologous genes were also detected in mice, primates and some other species (8, 9).