The human GARS-AIRS-GART gene encodes two proteins which are differentially expressed during human brain development and temporally overexpressed in cerebellum of individuals with Down syndrome
The human GARS-AIRS-GART gene encodes two proteins which are differentially expressed during human brain development and temporally overexpressed in cerebellum of individuals with Down syndromeGary Brodsky1,*, Tristan Barnes1, John Bleskan1, Laurence Becker2, Marlin Cox1 and David Patterson1,3
1Eleanor Roosevelt Institute, 1899 Gaylord Street, Denver, CO 80206, USA, 2Department of Pathology, The Hospital for Sick Children, Toronto, Ontario, Canada and 3Human Medical Genetics Program and Department of Biochemistry, Biophysics and Genetics and Medicine, University of Colorado Health Science Center, Denver, CO 80262, USA
Received May 16, 1997;Revised and Accepted August 9, 1997
Purines are critical for energy metabolism, cell signalling and cell reproduction. Nevertheless, little is known about the regulation of this essential biochemical pathway during mammalian development. In humans, the second, third and fifth steps of de novo purine biosynthesis are catalyzed by a trifunctional protein with glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART) enzymatic activities. The gene encoding this trifunctional protein is located on chromosome 21. The enzyme catalyzing the intervening fourth step of de novo purine biosynthesis, phosphoribosylformylglycineamide amidotransferase (FGARAT), is encoded by a separate gene on chromosome 17. To investigate the regulation of these proteins, we have generated monoclonal and/or polyclonal antibodies specific to each of these enzymatic domains. Using these antibodies on western blots of Chinese hamster ovary (CHO) cells transfected with the human GARS-AIRS-GART gene, we show that this gene encodes not only the trifunctional protein of 110 kDa, but also a monofunctional GARS protein of 50 kDa. This carboxy-truncated human GARS protein is produced by alternative splicing resulting in the use of a polyadenylation site in the intron between the terminal GARS and the first AIRS exons. The expression of both the GARS and GARS-AIRS-GART proteins are regulated during development of the human cerebellum, while the expression of FGARAT appears to be constitutive. All three proteins are expressed at high levels during normal prenatal cerebellum development while the GARS and GARS-AIRS-GART proteins become undetectable in this tissue shortly after birth. In contrast, the GARS and GARS-AIRS-GART proteins continue to be expressed during the postnatal development of the cerebellum in individuals with Down syndrome.
Purine biosynthesis plays an essential role in a number of different cellular processes. Purine nucleotides function as precursors for RNA and DNA synthesis, coenzymes, energy transfer molecules and regulatory factors in higher organisms. Two metabolic pathways for purine biosynthesis exist in humans. One pathway is responsible for the de novo synthesis of purine bases while a second salvage pathway is responsible for the re-utilization of purine bases. Considering the importance of purine nucleotides to cellular energy metabolism, reproduction and signalling, it seems likely that purine synthesis would be subject to genetic and/or biochemical regulation resulting in developmental, tissue and cell type specific regulation.
The human GARS-AIRS-GART locus, located on chromosome 21 (1 ), encodes three of the 10 enzymatic steps necessary for the conversion of phosphoribosyl pyrophosphate to inosine monophosphate by the de novo purine pathway. The three enzyme activities are encoded in a linear, non-overlapping fashion on the GARS-AIRS-GART mRNA (2 ). Starting at the 5' end of the cDNA, the three enzyme activities are glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). These enzymatic activities catalyze the second, fifth and third step of the de novo purine pathway, respectively. The fourth enzyme in the pathway, phosphoribosylformylglycineamide amidotransferase (FGARAT), is encoded by a separate gene on chromosome 17 (3 ).
While the monofunctional GARS, AIRS and GART genes found in microorganisms (4 ) appear to have evolved into a single GARS-AIRS-GART gene encoding a multifunctional GARS-AIRS-GART protein in Drosophila, chicken, mouse and human, analysis of RNA from each of these species indicates the presence of two classes of transcripts (2 ,5 ,6 ). One of these messages encodes a full length trifunctional protein while the other encodes only the GARS protein domain.
The studies described here were designed to begin an assessment of the roles of GARS, GARS-AIRS-GART and FGARAT in the regulation of purine synthesis. We report the generation of antibodies which specifically recognize the GARS, AIRS and GART domains of the human GARS-AIRS-GART protein. Western blot experiments performed on protein extracts from human tissue and cultured cells demonstrate the existence of a separate human GARS protein. Expression studies and sequence analysis indicate that both the GARS and GARS-AIRS-GART messages are transcribed from the human GARS-AIRS-GART gene. This is accomplished by alternative use of a splice donor site located at the 5' end of the intron separating the last GARS exon from the first AIRS exon. Western blot analysis of pre- and postnatal human cerebellum protein extracts demonstrates that both the GARS and GARS-AIRS-GART proteins are developmentally regulated in this tissue, with both proteins being primarily expressed during prenatal and early postnatal development. The relative expression of the GARS and GARS-AIRS-GART protein changes dramatically during this time and the levels of both proteins are greatly reduced within approximately one month of birth. The use of our previously reported anti-FGARAT antibody demonstrates that the level of this protein does not change in this tissue during the developmental period studied.
Because individuals with Down syndrome have elevated serum purine levels (7 ) and because elevated purine levels have been associated with mental retardation (8 ,9 ), we also examined the expression of GARS, GARS-AIRS-GART and FGARAT in the developing cerebellum of individuals with Down syndrome. These experiments demonstrate that the down-regulation of the GARS-AIRS-GART protein is much less pronounced in this brain region in individuals with Down syndrome and that expression of the GARS protein is constitutive in the trisomic tissue samples examined.
Monoclonal and polyclonal antibodies were generated against human GARS, AIRS and GART fusion proteins (see Materials and Methods). Western blot analysis of human HepG2 and HT1080 tissue culture cell protein extracts demonstrates that human cells express both a full length GARS-AIRS-GART protein as well as a smaller GARS protein (Fig. 1 ). The monoclonal antibody and the polyclonal antiserum raised against the GARS fusion protein both recognize a 110 kDa protein which is the expected size for the trifunctional GARS-AIRS-GART protein, as well as a 50 kDa protein which is the predicted size for a monofunctional GARS protein. The polyclonal antisera raised against the AIRS and GART fusion proteins only recognize the 110 kDa protein. Therefore, the 50 kDa peptide recognized by the GARS antibodies must contain only GARS protein sequences. The GARS-AIRS-GART protein expressed by the HepG2 cell line is larger than that expressed in HT1080 cells suggesting that this protein is alternatively processed or post-translationally modified in the HepG2 cell line.
To confirm that the antigenic species recognized by the antibodies are the GARS and GARS-AIRS-GART proteins, western blot analysis was performed on protein extracts from mutant CHO-K1 cell lines (Fig. 2 ). These mutant cell lines have been isolated on the basis of auxotrophy for purines and consequently require media supplemented with hypoxanthine for growth. The purine auxotrophy in the subset of mutants examined has been shown to be due to a lack of GARS, AIRS and/or GART enzymatic activities (Bleskan and Patterson, unpublished results) (Table 1 ) (10 ). The GARS monoclonal antibody (Fig. 2 A) recognizes a50 kDa GARS protein in addition to the 110 kDa GARS-AIRS-GART protein in the parental CHO-K1 cell line. The pattern of expression of the 110 kDa GARS-AIRS-GART protein, the 50 kDa GARS protein, and the relative amounts of these two proteins are altered from the parental CHO-K1 control in at least five of the six mutant cell lines.
To determine if the GARS-AIRS-GART gene encodes both the GARS and GARS-AIRS-GART proteins, a human clone was isolated and used for expression analysis. A human genomic P1 library was screened using a primer set which amplifies sequences surrounding the translation initiation codon of the GARS-AIRS-GART cDNA (Fig. 3 A). A P1 clone was isolated and analyzed by restriction enzyme digestion and CHEF analysis (Fig. 3 B). This P1 clone has an insert of 84 kb and contains a SalI restriction site located 10 kb upstream, and a NotI site located 15 kb downstream of the GART gene (11 ).
A PCR primer set was designed to amplify a 236 bp region of the putative GARS-AIRS-GART promoter region (Fig. 3 A). A second primer set was generated to yield a 149 bp amplimer from the 3' UTR of the GARS-AIRS-GART cDNA (Fig. 3 A). The isolated P1 clone produces the expected amplimers when used as the template for PCR reactions with these primer sets (Fig. 3 C).
The insert from the P1 clone was isolated from a preparative CHEF gel and transfected into the mutant J405 cell line. Transfectants were selected on media lacking purines. No revertants were obtained in the control transfections when the DNA insert was omitted. Protein extracts isolated from five independent transfectant clones grown in the absence of added purines were analyzed on a western blot (Fig. 4 ). The GARS MAb demonstrates an increase in the amount of full length GARS-AIRS-GART protein relative to the J405 parental strain in all the transfected cell lines. In addition, an increase in the expression of the 50 kDa GARS protein relative to the J405 control can be seen. Thus the 84 kb genomic insert in this P1 clone contains the sequence information necessary for expression of both the 110 kDa GARS-AIRS-GART protein and the 50 kDa GARS protein as well as the GARS, AIRS and GART enzymatic activities necessary to complement the purine auxotrophy of the J405 cell line.
Analysis of the mouse GART locus has shown that intronic polyadenylation signals within the GARS-AIRS-GART gene are used for expression of a separate GARS protein (15 ). To determine if a similar mechanism is responsible for the production of the human GARS and GARS-AIRS-GART proteins, the intron present between the last GARS exon and first AIRS exon was PCR amplified using primers which hybridize to coding regions 107 bp upstream and 117 bp downstream of the GARS-AIRS junction (Fig. 5 A). A 224 bp amplimer was obtained when the GARS-AIRS-GART cDNA was used as template whereas a 4.0 kb amplimer was produced in PCR reactions when the P1 clone containing the genomic GARS-AIRS-GART locus was used as template (Fig. 5 B).
To determine if the GARS and GARS-AIRS-GART proteins are differentially expressed or developmentally regulated, Western blot analysis was performed on equal amounts of total protein isolated from human brain cerebellum samples of various ages using the GARS MAb (Fig. 7 A), the AIRS polyclonal antiserum (Fig. 7 B) and a [beta]-actin MAb control (Fig. 7 C). The AIRS antiserum reveals the presence of the 110 kDa GARS-AIRS-GART protein in all of the prenatal brain samples and shows that it is reduced to barely detectable levels in the postnatal samples.
The western blot performed with the GARS monoclonal antibody demonstrates the same pattern of expression for the GARS-AIRS-GART protein as that seen with the AIRS polyclonal antiserum. In addition, the 50 kDa GARS protein is also evident in the prenatal and one day old samples, with the one day old sample providing the strongest signal. By 20 days, the GARS protein is reduced to a level at which it is barely detectable. The [beta]-actin western blot demonstrates that approximately equal amounts of total protein are present in each lane. A western blot performed on the same protein samples using an antibody specific for FGARAT (3 ) demonstrates that there is little or no change in the level of this protein in any of these samples (Fig. 8 ).
The development of the cerebellum is altered in individuals with Down syndrome (17 ). Additionally, individuals with Down syndrome have elevated purine levels (7 ), and elevated purine levels caused by mutations in purine metabolic enzymes have been associated with mental retardation and sensorineural deafness (8 ,9 ,18 ). Therefore, the levels of the GARS, GARS-AIRS-GART and FGARAT proteins in the developing cerebellum of individuals with Down syndrome were examined. Figure 9 shows that, in contrast to the expression patterns seen in normal postnatal cerebellum, the GARS and GARS-AIRS-GART proteins continue to be expressed until at least day 49 after birth in the cerebellum of individuals with Down syndrome. Again, the levels of FGARAT do not change significantly during this developmental period (Fig. 9 C). In addition, while the expression of the GARS-AIRS-GART protein decreases with age in the postnatal Down syndrome cerebellum samples (Fig. 9 A and B), no reduction in GARS expression is observed (Fig. 9 A).
We demonstrate developmental regulation of the trifunctional GARS-AIRS-GART protein and the monofunctional GARS protein in human cerebellum (Fig. 7 ). Furthermore, we demonstrate aberrant regulation of the levels of these proteins in the cerebellum of individuals with Down syndrome.
Purine biosynthesis is a critical biochemical pathway. In spite of this, little is known about the tissue specific or developmental regulation of the enzymes in this pathway, or the genetic regulation of the genes encoding these enzymes. The trifunctional GARS-AIRS-GART protein contains the second, third and fifth enzymatic activities of the 10 step pathway leading to IMP synthesis. In contrast to the observed developmental regulation of GARS-AIRS-GART and GARS, expression of FGARAT, which catalyzes the fourth step in this pathway, appears to be constitutive (Fig. 8 ). This suggests a role for the GARS and GARS-AIRS-GART proteins in the developmental regulation of purine synthesis.
We have hypothesized that trisomy of the GARS-AIRS-GART gene in Down syndrome might cause the elevated purine levels observed in these patients, and that this elevation might be related to the mental retardation and sensorineural deafness seen in individuals with Down syndrome (19 ,20 ). While both the GARS and GARS-AIRS-GART protein become undetectable in normal cerebellum between 1 and 20 days after birth (Fig. 7 ), both of these proteins continue to be expressed in the cerebellum of individuals with Down syndrome at least until day 49 after birth (Fig. 9 ). These results indicate that the elevated purine levels observed in patients with Down syndrome may be due to GARS and/or GARS-AIRS-GART overexpression.
The cerebellum is known to develop aberrantly in individuals with Down syndrome (17 ). It is not clear at this time whether the altered GARS and GARS-AIRS-GART protein levels are a part of the cause of this aberrant development or a consequence of it.
Our understanding of the genetic regulation of the GARS-AIRS-GART locus is complicated by the production of an alternative transcript which encodes only the GARS enzymatic domain (Fig. 2 ). This protein is produced by differential use of an intronic polyadenylation signal located in the intron separating the last GARS exon from the first AIRS exon (Fig. 6 ). Separate GARS and GARS-AIRS-GART mRNAs have been observed in human (2 ), mouse (6 ), chicken (2 ) and Drosophila melanogaster (5 ). In human, mouse and Drosophila, the GARS message contains an in-frame stop codon consisting of the TAA present as part of the 5' splice donor site. This results in the expression of a GARS protein which is identical in amino acid composition to the GARS domain in the trifunctional protein.
A number of genes have been identified in which the alternative use of an intronic polyadenylation signal has been shown to provide a mechanism for tissue-specific or developmental regulation of protein expression. The most thoroughly characterized of these are the immunoglobulin heavy chain genes (21 ). In this case the alternative use of an intronic polyadenylation signal provides a mechanism for developmental switching between an excreted form of IgM and a membrane bound form during B cell maturation (22 ).
Both the overall levels of expression and the relative amounts of the GARS and GARS-AIRS-GART proteins are developmentally regulated in human cerebellum (Fig. 7 ). The trifunctional GARS-AIRS-GART protein is expressed at higher levels in the prenatal brain samples versus the postnatal samples. In addition, the GARS-AIRS-GART protein is present at equal or greater levels than the GARS protein in the prenatal brain samples whereas the GARS protein is the predominant protein species in the 1 day old postnatal brain sample. Remarkably, the expression of both the GARS and GARS-AIRS-GART protein is greatly reduced in the 20 and 66 day old postnatal samples suggesting a dramatic change from the de novo to the salvage purine pathway around the time of birth.
A developmental change from the de novo to the salvage purine pathway is consistent with the observation that the salvage pathway is the major source of purine nucleotides in differentiated mammalian cells (23 ). The relatively high levels of GARS-AIRS-GART expression in the prenatal versus postnatal tissues suggests that the de novo pathway may be the major source of purine nucleotides during human embryonic cerebellum development.
Three oligonucleotide primer sets containing EcoRI or BamHI restriction sites were synthesized for polymerase chain reaction (PCR) amplification and subcloning of the GARS, AIRS and GART coding domains (Fig. 10 A). The GARS domain primer set amplifies nucleotides 1-1299, the AIRS domain primer set amplifies nucleotides 1300-2427 and the GART domain primer set amplifies nucleotides 2428-3030 of the human GARS-AIRS-GART cDNA.
Normal human cerebellum samples were obtained from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland at Baltimore. Protein isolations were performed as previously described (3 ). Protein concentrations were determined using the BioRad Microassay reagent. Equal amounts of total protein were resolved by discontinuous SDS-polyacrylamide gel electrophoresis. Immunoblotting and detection were performed as previously described (3 ). Equal protein loading was confirmed by Ponceau S staining of the undeveloped blots. All western blot experiments were repeated a minimum of three times.
A cosmid clone, cosGART16 (11 ) containing the 5' end of the human GARS-AIRS-GART gene was CsCl purified and directly sequenced from the 5' end of the GARS coding region into the 5' untranslated region (UTR). Subsequent primers were then generated from the DNA sequences obtained.
A human genomic P1 library (29 ) was screened using standard PCR conditions with a pair of oligonucleotide primers which amplify nt -160 through +98 of the human GARS-AIRS-GART cDNA. The isolated P1 clone was further analyzed using two PCR primer sets. The first primer set amplifies the region from 77 to 312 nt upstream of the start of the GARS-AIRS-GART cDNA in the putative GARS-AIRS-GART promoter region (5' primer, TCCCTGCCCCATTGGTC; 3' primer, TAGCTGCTCAGCTCTGA). The second primer set amplifies nt 3016-3164 which extends across the stop codon and into the GARS-AIRS-GART 3' UTR. Analytical restriction digests of P1 DNA were resolved on a CHEF DR II Pulsed Field Electrophoresis gel apparatus (Bio-Rad Laboratories, Inc.) using 1% FastLane agarose (FMC BioProducts). The P1 insert was isolated from a preparative 1% SeaPlaque agarose (FMC BioProducts) CHEF gel (30 ).
The isolated P1 insert was transfected into the CHO J405 (Ade-PCG) (31 ) cell line using Lipofectin reagent (Gibco-BRL). Cells were plated at 2 * 105 cells/100 mm tissue culture plate in F12 medium supplemented with 5% fetal calf serum. After 24 h the media was removed and the plates were rinsed and resuspended in F12 without added serum. A total of 2 [mu]g of insert DNA in 50 [mu]l of Lipofectin reagent was added slowly to the cells and the plates incubated for 16 h. An equal volume of F12 supplemented with 10% fetal calf serum was added and the plates incubated an additional 24 h. The cells were then rinsed twice with saline G, and F12 without hypoxanthine was then added for selection of prototrophic transfectants.
PCR primers incorporating EcoRI restriction sites which amplify nt 1183-1406 of the GARS-AIRS-GART cDNA were synthesized. Using standard PCR conditions the primers produce the expected 224 bp amplimer when the GARS-AIRS-GART cDNA is used as template (data not shown). The Expand Long Template PCR kit (Boehringer Mannheim) was used with these same primers and the human GARS-AIRS-GART containing P1 clone as template. The resulting PCR amplimer was purified, digested with EcoRI and subcloned into pBS-SK(+). The desired clone was identified by restriction enzyme analysis and the insert was sequenced from the 5' end of the GARS domain. T3 and T7 universal primers were used as the initial sequencing primers. Subsequent primers were generated from the previously obtained DNA sequences.
The authors wish to thank Lynda Fox, Miles Brennan and Katheleen Gardiner for their comments and suggestions. We also wish to thank Sally Wisniewski, Robert Vigorito, and H. Ronald Zielke at the Brain and Tissue Bank for Developmental Disorder at the University of Maryland at Baltimore for their help in obtaining the human tissue samples. This is contribution #1613 from the Eleanor Roosevelt Institute and the Thomas G. and Mary Vessels Laboratory for Molecular Biology. This work was supported by NIH grants AG00029, HD17449 and CA46934.
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*To whom correspondence should be addressed. Tel: +1 303 333 4515; Fax: +1 303 333 8423; Email: garyb@eri.uchsc.edu
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