2 Department of Molecular, Cellular, and Craniofacial Biology, School of Dentistry and Departments of Biochemistry and Molecular Biology and Pharamacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40292
3 Biological Chemistry Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Received on January 19, 2003; revised on February 25, 2003; accepted on February 26, 2003
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Abstract |
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Key words: glycosyltransferases / mucins / O-linked glycosylation / real-time PCR
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Introduction |
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Despite the considerable investigative effort expended concerning the size and expression patterns of the ppGaNTase family, so far no quantitative comparison of the levels of all ppGaNTase isoforms in any tissue of any species has been made. Such a comparison cannot be made by enzyme activity assay because substrate specificities of the isoforms are largely overlapping. Within a cell the ppGaNTase activity for any given substrate will be the sum of the activities for all isoforms present, and the contribution from any one isoform cannot be determined. Therefore, in the present article we determined the expression of ppGaNTase isoforms at the level of mRNA by real-time polymerase chain reaction (PCR) using the Taqman method. The results demonstrate that many tissues share a common pattern of having isoforms T1 and T2 as the most highly expressed family members, whereas a few other tissues have expression patterns unique to those tissues. One putative isoform designated here as isoform Tc is expressed only in the testis. Finally, the expression of several isoforms varies during pregnancy and lactogenesis in mammary tissue.
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Results |
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Comparison among mouse tissues of ppGaNTase isoform expression
As described in the Discussion, we used glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as internal reference for all of the real-time PCR results reported here despite the fact that GAPDH expression varies under certain circumstances and may also vary between tissues. The following comparisons of expression levels among tissues must be viewed with that potential caveat in mind. Isoforms T1 and T2 were expressed in all tissues tested (Figure 1), confirming the ubiquitous expression found in earlier northern blotting studies and in recent real-time PCR analyses of isoform T1 in human tissues (Zhang et al., 2003). However, the expression of isoform T1 differed widely among tissues of group A with the expression in spleen being more than 10-fold greater than that in heart, muscle, or thyroid. Interestingly, isoforms T1 and T2 were the most strongly expressed isoforms only in group A tissues. The highest expression of any isoform in any tissue was that of isoform T3 in testis.
Recently isoform T13 was cloned, shown to be expressed most strongly in human nervous tissue, and found to synthesize Tn antigen (Zhang et al., 2003). Similarly, we detected isoform T13 expression most strongly in brain (1/fold expression=8.6x10-4; Figure 1), whereas the levels in lung and colon were 8-fold and 42-fold lower, respectively, than in brain, and the level in the other tissues was undetectable. Interestingly, the level of isoform T13 in brain was actually very low compared to the expression of the other isoforms; that is, isoform T13 ranked ninth when the isoforms were ranked from strongest to weakest, and the level of the isoform most strongly expressed in the brain, T2, was 22-fold greater than isoform T13. Nevertheless, this example illustrates the point that the expression of an individual isoform that bears a particular enzymatic specificity may be of functional importance (Zhang et al., 2003
) even if its actual level of expression is quite low.
Adding the individual expression values for each isoform produces a value for the total expression of all ppGaNTase isoforms in each tissue. These total expression values differed greatly among tissues. The strongest total expression was found in testis (1/fold expression=0.47) followed by sublingual gland (0.31), lung (0.24), spleen (0.18), prostate (0.15), and colon (0.1). With the exception of the spleen, these tissues with strong total expression either had glandular components or contained actively secreting cells, such as the goblet cells of the lung and colon. Among the tissues with lower total expression, all were in group A, all had T1 and T2 as the most strongly expressed isoforms, and none were glandular except for the thyroid.
Expression patterns in mammary tissue
The preceding analysis of constitutive ppGaNTase isoform expression among mouse tissues is important for establishing baseline patterns and for identifying tissue-specific isoforms. However, additional information relevant to isoform function can be gained by comparing isoform expression under different physiological conditions. As an example of this approach, we analyzed mammary tissue because of its robust mucin production and because of the major changes that occur during pregnancy and lactation. The pattern in virgin mammary gland (Figure 2) was similar to that found in the group A tissues, with the two most active isoforms being isoforms T1 and T2 and with the next most prominent isoform being T8 as in the lung. During pregnancy, the expression of isoform T3 increased steadily, reached a final value at day 19 that was 6.9-fold greater than the virgin level and then decreased toward the virgin level during lactation and involution. Isoform T10 increased to a lesser extent during pregnancy and then continued to increase during lactation so that by day 10 of lactation the level was 7.7-fold greater than virgin level. During involution T10 expression also decreased toward the virgin level.
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Comparison of ppGaNTase isoform expression among mouse strains
The preceding data were obtained with tissues from several strains of mice (see Materials and methods). Therefore, we sought to determine if there were differences in expression of ppGaNTase isoforms among mouse strains. For this purpose we analyzed the expression in kidneys from representative strains which differ at the major histocompatibility complexnamely, Balb/c (H2-d), C3H (H2-k), and C57/BL (H2-b). As with many other tissues described, isoform T1 was the most highly expressed isoform in the kidney (Figure 1). However, the most distinctive feature of ppGaNTase expression in the kidney was that isoform T11 was the second most highly expressed isoform. This finding is consistent with the recent report of Schwientek et al. (2002), who found T11 to be most highly expressed in both human and mouse kidney among tissues screened by northern blotting. Isoform T11 was also expressed in lung and prostate about fivefold less strongly than in kidney (Figure 1).
Interestingly, the only statistically significant difference in isoform expression between strains was for isoform T11 between Balb/c and C57/BL mice (Table I). Expression of T11 was almost twice as great in the Balb/c kidneys as compared to C57/BL (Table I), whereas the level in C3H kidneys was intermediate between the other two strains. This difference between strains was apparent in kidneys from both males and females. The causes of these strain-specific differences in T11 expression remain to be determined. In summary we conclude that the expression of the ubiquitously expressed isoforms 1 and 2 (Figures 1 and 2) does not vary between strains nor does the expression of the strongly expressed isoforms T3, T8, and T14. However, the example of isoform T11 indicates the need for future studies of other tissues to test for strain differences in expression of the other isoforms that are not prominent in the kidney and to determine the causes for the strain-specific expression of isoform T11.
A striking finding of the analysis of isoform expression in the kidney was that the expression of isoform T1 differed depending on gender (Table I). In all three strains expression was stronger in males than in females although the difference was statistically significant only in the C3H strain and when the data from all three strains were combined (Table I). The basis for these gender-specific differences in expression will require additional analysis, but a precedent may be the androgen responsiveness of mouse kidney beta-glucuronidase (Thornton et al., 1998). However, because other tissues and RNAs used in the present study were pools from both males and females, except for the gender-specific tissues, this gender difference was not pursued in the present study.
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Discussion |
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One striking finding is the contrast between the large group of tissues, known as group A here, that exhibit a relatively simple pattern of isoform expression and the few tissues, namely, colon, kidney, sublingual gland, and testis, with a much more complex pattern (Figure 1). The common pattern of group A tissues is that isoforms T1 and T2 are the predominant isoforms (Figure 1). These two isoforms have been proposed to serve "housekeeping" ppGaNTase functions partly because of their ubiquitous expression. However, immunohistochemistry with isoform-specific antibodies revealed that T1 and T2 were expressed in connective tissue cells of some tissues (Mandel et al., 1999), raising the possibility that ubiquitous expression by RNA analysis of whole organs may be due to cell-specific expression within cells found in all tissues. This point illustrates two limitations of real-time PCR analysis of isoform expression in tissues. First, assays of tissues do not reflect the relative expression profiles in specific cell types, thus necessitating the localization of expression within tissues by in situ hybridization. Second, demonstration of the actual production of the protein product must be revealed by immunohistochemistry. Both of these lines of study will be pursued in future investigations.
The complex patterns of isoform expression in colon, kidney, sublingual gland, and testis may reflect the complex architecture of those tissues with certain isoforms being expressed in different cell types. An additional or alternative explanation may be the need in those tissues for the synthesis of a variety of O-linked glycans. The fine structural specificity of the ppGaNTase isoforms is only partially understood (Hassan et al., 2000; Ten Hagen et al., 2003
). In contrast to isoforms T1 and T2, which accept a large variety of substrates, isoforms T7 and T10 glycosylate glycopeptide substrates but not nonglycosylated substrates (Ten Hagen et al., 1999
, 2001
). Interestingly, both of these isoforms are elevated in colon and sublingual gland. Also, several isoforms prefer substrates of defined amino acid sequence (Hagen et al., 1997
; Bennett et al., 1998
, 1999
; Ten Hagen et al., 1998
), and certain substrates may require a particular isoform for their glycosylation (Iwasaki et al., 2003
; Zhang et al., 2003
). Thus, it is not surprising that tissues with ample mucin production, such as colon, sublingual gland, and testis, might require high-level expression of multiple ppGaNTase isoforms. However, additional data on the precise specificities of all isoforms must become available before we can fully interpret the results of isoform expression reported here.
Genetic ablation of individual ppGaNTase isoforms in mice so far has not produced recognizable phenotypes (reviewed in Ten Hagen et al., 2003). This failure may be due to functional redundancy among isoforms, suggesting that the knockout of multiple isoforms may be necessary. In this regard the real-time PCR results may be very useful because they provide a picture of relative expression levels of isoforms not previously available. For example, isoforms T1 and T2 constitute nearly all of the ppGaNTase expression in some group A tissues (Figure 1). Therefore, these findings lead to the prediction that a double knockout of T1 and T2 may answer the question as to whether ppGaNTase activity is essential in those tissues for viability and/or for proper development. The isoform expression results should also be useful during the analysis of knockout mice because the data focus attention onto those tissues showing strongest expression of each isoform.
Similarly, the results also identify the potential tissue distribution of several putative isoforms. The most obvious example is putative isoform Tb, which is expressed strongly and exclusively in the testis. Putative isoform Ta is expressed most strongly in brain, lung, and testis and putative isoform Tc in lung and ovary. Future efforts will be directed at determining which of these putative isoforms encode functional enzymes.
All expression data are based on the difference of each experimental value from the value for the internal reference, GAPDH, in that tissue. Although GAPDH is frequently used for this purpose, its expression is dependent on numerous factors (see reviews Suzuki et al., 2000; Bustin, 2001
) and therefore may vary among tissues. As one means of assessing the present results independently of GAPDH, we compared our real-time PCR results with published northern blot results. There was generally a good correlation between the two data sets for isoforms 3 (Zara et al., 1996
), 4 (Hagen et al., 1997
), 5 (Ten Hagen et al., 1998
), 7 (Ten Hagen et al., 1999
), 10 (Ten Hagen et al., 2001
), 11 (Schwientek et al., 2002
), 12 (Guo et al., 2002
), 13 (Zhang et al., 2003
), and 14 (Wang et al., 2003
). For isoforms T1 and T2 northern blot analysis showed ubiquitous expression in rat and mouse tissues (Hagen et al., 1997
; Ten Hagen et al., 2001
), thus making it difficult to compare those results quantitatively with the present real-time PCR data. Northern analysis data for human isoform T6 (Bennett et al., 1999
) and putative human isoform T8 (White et al., 2000
) did not correlate with the present real-time PCR data.
The results of isoform expression in mammary glands (Figure 2) provide an example of how physiological conditions may influence the expression profile of ppGaNTase isoforms. The virgin mammary gland consists of adipocytes with the ductal epithelium dispersed through the fat pad (reviewed in Hennighausen and Robinson, 2001; Shillingford and Hennighausen, 2001
). During pregnancy the epithelium proliferates and differentiates under the influence of progesterone, placental lactogens, and prolactin. During lactation the ducts form side branches under the continued influence of prolactin, resulting in the secretory epithelium's completely filling the fat pad. Finally, when suckling ceases, the epithelium undergoes apoptosis and tissue remodeling, a process that is reversible at day 1 and irreversible by day 4 of involution.
The results indicate that the expression of ppGaNTase isoform T3 increased during pregnancy and isoform T10 increased during both pregnancy and lactation, whereas isoform T2 decreased during pregnancy and putative isoform T8 decreased during both pregnancy and lactation. At present it is not known whether the expression of any of these four isoforms is regulated by hormones present in mammary tissue. An alternative to hormonal regulation of isoform expression is that isoform T2 is expressed in the epithelial cells, whereas isoform T8 is expressed in the adipocytes, and, therefore, their changes in level of expression simply reflect the nature of the mammary tissue at each stage. Information about the regulation of ppGaNTase gene expression is available only for isoform T3. Nomoto et al. (1999) found that T3 expression is regulated by multiple systems, including transcription factor binding sites and a stem-and-loop structure in epithelial gland cells. Thus, future studies will be directed at assessing the factors responsible for these dramatic changes in ppGaNTase isoform expression during mammary gland development.
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Materials and methods |
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Quantitative mRNA analysis by real-time PCR
mRNA gene expression levels were determined after reverse transcription by real-time PCR using an Applied Biosystems PRISM 7700 Sequence Detection System (Foster City, CA). Total RNA was treated with DNase, and then cDNA was synthesized using random hexamers and the SuperScript first-strand synthesis kit (Invitrogen). Primers and fluorogenic probes were designed using Primer Express (version 1.5, Applied Biosystems) according to the rules of Livak (1999). Because of large regions of homology among the members of the ppGaNTase family (Hagen et al., 1999
), primer-probe sets were chosen in regions of low homology to avoid cross-reactions with other isoforms. Regions of homology were identified by ClustalX. Primers were prepared by G. Poy, NIDDK, NIH, or were purchased from MWG Biotech (High Point, NC). Probes were purchased from Geneprobe Technologies (Gaithersburg, MD).
All assays were performed with the single thermocycling protocol of 50°C, 2 min; 95°C, 10 min; and 40 cycles of 95°C, 15 s, followed by 60°C, 1 min. The amplification efficiency was determined for each primer-probe set (Table II) as previously described (Bustin, 2001). Briefly, Ct values were determined for a series of 10-fold dilutions of template and the results plotted as the log of the initial target copy number versus Ct. All sets produced slopes close to the theoretical ideal of -3.3. The set of primers and probe for the internal reference, GAPDH, was purchased from Applied Biosystems.
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Taqman results produce a Ct (threshold cycle) at which the fluorescence produced by cleavage of the fluorogenic probe rises above a background (or so-called threshold) level. Experimental values were compared to those obtained for a primer-probe set specific for an internal standard, GAPDH, and a delta Ct value was obtained that was the difference in the Ct values between experimental and GAPDH. In all cases expression of GAPDH was greater than that of any ppGaNTase isoforms. Because each cycle is actually a doubling, the delta Ct values were converted to a fold difference between experimental and GAPDH (2 cycles=4-fold, 3 cycles=8-fold, etc.). The data are presented as 1/(fold difference) so that stronger expression is represented by a higher value in Figures 1 and 2.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: wwyoun01{at}gwise.louisville.edu
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Abbreviations |
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References |
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