Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, FL 33176, USA
Received on August 5, 2002; revised on November 26, 2002; accepted on December 5, 2002
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Abstract |
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Key words: 5' untranslated region / alternative splicing / glycosyltransferase / mRNA
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Introduction |
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One important regulatory step in the synthesis of O-linked oligosaccharides is the conversion of a core 1 oligosaccharide structure to a core 2 structure by addition of a N-acetylglucosamine (GlcNAc) in a ß1,6 linkage to the N-acetylgalactosamine (GalNAc) residue attached to the serine or threonine in the protein chain. The ß1,6 branched GlcNAc provides the requisite substrate for further sugar additions by other glycosyltransferases. Many biologically significant oligosaccharide structures are constructed on this branch, including some important cell recognition and adhesion molecules.
The core 2 branch can be added by at least three enzymes, the core 2 ß1,6 N-acetylglucosaminyltransferase (C2GnT-L) L-type for leukocyte, the C2GnT M-type for mucin (Yeh et al., 1999), and the C2GnT T-type for thymus (Schwientek et al., 2000
). The T-type is expressed almost exclusively in the thymus, whereas the M-type is expressed predominantly in mucin-producing cells (Hiraoka et al., 2000
). The L-type, also referred to as core 2 ß1,6-N-acetylglucosaminyltransferase I (C2GnT I) is more widely expressed in many tissues, including mucin-producing cells and lymphocytes. C2GnT I expression is regulated in T lymphocytes as they mature. Immature T cells must down-regulate C2GnT I as they transit through the thymus because T cells expressing the core 2 branch in the thymus will be induced to apoptose by binding to galectin-1 (Baum et al., 1995
; Galvan et al., 2000
). However, when T cells or B cells become activated (Piller et al., 1988
; Nakamura et al., 1998
), C2GnT I activity must be induced because the expression of the core 2 branch is critical for the synthesis of selectin adhesion molecules required for leukocyte recruitment to sites of inflammation (Cho and Cummings, 1994
; Rosen and Bertozzi, 1994
; Wilkins et al., 1996
; Hemmerich et al., 1995
).
Improper expression of C2GnT I is associated with a number of pathological conditions (Tsuboi and Fukuda, 2001). C2GnT I gene knockout mice are moderately neutrophilic and show abnormal lymphocyte trafficking (Ellies et al., 1998
; Sperandio et al., 2001
). The overexpression of C2GnT I is associated with T cell leukemias (Saitoh et al., 1991
) as well as colon and other cancers (Brockhausen et al., 1991b). Some immunodeficiencies have been associated with elevated expression of C2GnT I, such as Wiskott-Aldrich syndrome (Datti et al., 1994
) and HIV type 1-infected T cells (Lefebvre et al., 1994
). It has been suggested that elevated expression of C2GnT I leads to myocardial dysfunction in diabetic rats (Nishio et al., 1995
) and transgenic mouse models (Koya et al., 1999
). These findings indicate that the expression of C2GnT I is important for many normal cellular activities and that aberrant expression is associated with various pathological conditions.
The C2GnT I gene provides an interesting model for the regulation of gene expression. It is expressed in many but not all cell types in housekeeping-like fashion. It is also expressed in a developmentally regulated manner in T cells and mucin-producing epithelial cells, two cell types of different embryological origin. In addition, the expression of this gene is associated with many types of malignant cancers. Previous reports have indicated that there are multiple promoters for the mouse C2GnT I gene that are used tissue specifically (Sekine et al., 1997). Therefore, this study was initiated to investigate the human gene and identify the regulatory elements in the C2GnT I gene that are important for its transcription. We identify four different transcription initiation sites that are differentially used by different tissue types. Furthermore, there is also a tissue-specific, alternative exon use in the 5' untranslated region (UTR) for a total of 5 forms of the C2GnT I mRNA.
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Results |
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One possible problem with using 5' RACE to identify the 5' end of an mRNA is the potential for premature termination of cDNA synthesis by the reverse transcriptase prior to reaching the actual 5' terminus. If this were to occur, the actual transcription initiation site would be further upstream of the observed RACE sequence. Premature termination could be caused by secondary structure in the mRNA and/or regions of high GC content. The cDNA panel used for these experiments is prepared by a dual-cycling cDNA synthesis procedure that significantly reduces this problem. The cDNA is synthesized in two steps. The first step uses mouse mammary tumor virus reverse transcriptase at 42°C and is followed by an additional synthesis step using Tth DNA polymerase, a thermostable polymerase that also has reverse transcriptase activity, at 75°C. The use of high temperature during cDNA synthesis enhances the reverse transcription through RNA secondary structures. In addition, because there was a region of high GC content near the start of exon B, two additional experiments were done to confirm this as the 5' end of the mRNA. First, additional gene-specific primers complementary to sequences within exon B were made and used for another 5' RACE. No additional RACE products could be generated. In addition, as an alternate approach to confirm the 5' RACE results, another RACE protocol, RLM RACE, was used. This protocol is based on the presence of a 5' cap structure on the mRNA to give a RACE product. This procedure identified a capped mRNA with the same start site as previous 5' RACE results, strongly suggesting exon B as a transcription start exon.
Exon utilization in different tissues
To establish the extent and distribution of exon usage among the different tissues, primers were designed from each of the initial exons (i.e., exon specific primers) and were used as 5' primers in conjunction with Core2GSP2, the primer used for RACE, as the 3' primer to amplify cDNA from each of the tissues. The results of these experiments are shown in Figure 4 and are compiled in Table I. None of the 5' terminal sequences have been observed in combination via splicing with another 5' terminal exon. For example, exon A was never seen spliced to exons B or D in any of the RACE clones, as might be expected if exons B or D were not primary transcribed exons. This suggests that each of these exons represents initiation of transcription from an independent promoter.
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Alternative splicing was observed in a tissue specific fashion. Exon C has only been observed in transcripts containing exon B. Three tissues (heart, spleen, and liver) always splice from exon B to exon C. Ten tissues (placenta, pituitary, adrenal, pancreas, ovary, uterus, prostate, peripheral blood lymphocytes, fetal liver, and fat) appear not to splice exon C. The four tissues from the digestive and respiratory systems (colon, lung, small intestine, and stomach) show both types of messages. There appears to be an mRNA splice variant that is unique to testis. The testis mRNA shows two exon Dcontaining transcripts. One has the predicted size for an RNA spliced from exon D to exon E; the other is about 100 bases longer and may indicate another, not yet characterized exon. We have not cloned and sequenced this amplification product. These experiments show that the expression of the C2GnT I mRNAs is varied from tissue to tissue. It is possible that the presence of different cell types within each tissue is responsible for some of this complexity, but this is yet to be determined.
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Discussion |
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The existence of multiple promoters has been reported for a number of glycosyltransferase genes. The ß1,4 galactosyltransferase gene has two different transcription initiation sites within about 200 bases of each other that produce two mRNAs (Rajput et al., 1996). Other glycosyltransferases that have multiple promoters include N-acetylglucosaminyltransferases III (Koyama et al., 1996
) and V (Buckhaults et al., 1997
; Saito et al., 1995
) and
2,3-sialyltransferases I and IV (Kitagawa et al., 1996
). The C2GnT I gene is very similar to the
2,6 sialyltransferase I (SIAT1) gene. The SIAT1 gene is transcribed from three different promoters with B cell- and liver-specific promoters. It is also alternatively spliced in the 5' UTR (Lo and Lau, 1996
, 1999
). Interestingly, both the C2GnT I and SIAT1 genes have an exon that is common to all mRNAs that can be transcribed from its own upstream promoter or inserted into the mRNA by splicing. These similarities are quite striking and suggest some functional significance to the organization of these genes and the expression of 5' UTR sequences.
The existence of multiple promoters for the C2GnT I and other glycosyltransferase genes may be due to the need for the expression of this gene in many but not all cell types and/or at different times during embryogenesis. The individual promoters can be regulated independently so that the gene can be transcribed in different and overlapping cell types or in response to different signaling systems. Analysis of the promoter elements for each of these promoters should be able to sort out these differences. Another consequence of using different promoters is the addition of a different 5' UTR sequence to the mRNA. The 5' UTR sequences could affect translation or mRNA stability.
The expression of C2GnT I has been correlated with increased metastatic capacity (Yousefi et al., 1991) and is associated with a variety of malignancies. These include acute myeloblastic leukemia (Brockhausen et al., 1991a), colorectal cancer (Shimodaira et al., 1997
), pancreatic cancer (Beum et al., 1999
), pulmonary adenocarcinoma (Machida et al., 2001
), and tumors of the oral cavity (Renkonen et al., 2001
). The mechanism for the increased C2GnT I expression in these malignancies is not known. It is possible that malignant transformation induces C2GnT I expression by activating transcription from one or more of these promoters as previously seen with other glycosyltransferases (Chen et al., 1998
; Buckhaults et al., 1997
). The elevated expression of this gene is believed to promote metastasis, but the exact mechanism(s) have not been clearly elucidated. Because it is possible that reduction of the expression of this gene might have antimetastatic effects, it would be helpful to know whether one or more of these promoters is responsible. Further experiments are needed to examine the use of these promoters in different tumor cells to elucidate the mechanism of the expression of this gene in the various cancer cells.
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Materials and methods |
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To test for expression from each of the 5' exons, 5' exon- specific primers sequences within each of the exons were selected to be compatible for use with Core2GSP2 as a 3' primer. The sequences of these primers are: exon A, CTGGGACCAGAAAAGGTC; exon B, TGGGCATCCTCCTGAGACT; exon D, CACGGGAAGGAAGAACT; and exon E', ACATTAAGGGAAATGCTGC.
The cap-dependent RLM RACE kit was obtained from Ambion (Austin, TX), and used per the enclosed instructions. The same gene specific primers already described were used.
Oligonucleotide probe labeling and Southern blot hybridizations
Oligonucleotides used for hybridization were labeled with digoxygenin using the DNA 3' end labeling kit from Roche. The hybridizations were done using a 1 nM probe concentration and at 57°C. Samples were processed, and the hybridization was detected by binding with an alkaline phosphatase conjugated anti-digoxygenin antibody and chemiluminescence with CDP-Star also from Roche.
Cloning and sequencing amplification products
Amplification products were cloned using the pGEM T-easy vector system (Promega, Madison, WI). Sequencing was done using the DNA sequencing facility of the Sylvester Comprehensive Cancer Center at the University of Miami School of Medicine.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: nevis{at}miami.edu
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Abbreviations |
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References |
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