FORMACION PROFESIONAL Y TIPO DE VALORES

The structure and amount of pl,4-GalTase transcripts is regulated during murine spermatogenesis (Shaper, et al., 1990; Harduin, et al., 1992; Shaper, et al., 1994). The primitive germ cells, spermatogonia, only express the 4.1 kb mRNA transcript, which is identical to the 4.1 kb transcript found in most somatic cells (Shaper, et al., 1990). As the diploid spermatogonia enter meiosis and develop into pachytene spermatocytes, the expression of pl,4-GalTase reaches very low levels (Shaper, et al., 1990). Following differentiation to the haploid round spermatids the expression of the 4.1 kb transcript is replaced with higher levels of two shorter transcripts of 3.1- and 2.9 kb (Shaper, et al.,

1990). These truncated transcripts encode the same open reading frame as the 4.1 kb transcript but differ in their 5' and 3' untranslated regions (Shaper, et al., 1990). The shorter transcripts result from the use of alternative polyadenylation sites in the 3' untranslated region (Shaper, et al., 1990). The haploid round spermatids have an extra transcriptional start site (-732 bp) in the extended 5' untranslated region compared to spermatogonia and somatic cells (Shaper, et al., 1994).

Thus the murine pi,4-GalTase gene has been shown to have three transcriptional start sites: i) The distal one that is germ cell specific and found only in pachytene spermatocytes and early round spermatids (Shaper, et al., 1994); ii) The intermediate Start site used predominantly in almost all somatic cells and spermatogonia (Harduin, et al., 1992) and; iii) The proximal start site which is highly upregulated in mid- to late- pregnant and lactating mammary gland (Harduin^ et al., 1993). Further, there is some evidence to suggest that tissue-specific and housekeeping promoters are regulating the expression of the different transcripts (Shaper, et al., 1990; Harduin, et al., 1993; Rajput, et al., 1996).

Two pi,4-GalTase mRNA transcripts of 4.1 kb and 3.9 kb, coding for the long and short forms of pl,4-GalTase respectively, have been found in many tissues from different species (Shaper, et al., 1988a; Russo, et al., 1990; Shaper, et al., 1990). Pouncey, et al (1991) also identified the 4.1 kb transcript, sometimes with a smaller sized 3.0 kb transcript. Cells with varying levels of pl,4-GalTase expression were analysed for their transcriptional start sites (Harduin^ et al., 1993) and regulatory factors of transcription (Rajput, et al., 1996). Spl transcription factor bound to regions just upstream from the 4.1 kb start site (Rajput, et al., 1996). Different DNA-binding

proteins were located close to the 3.9 kb start sites including AP2 (in the lactating mammary gland) a mammary gland form of CTF/NFl, Spl and a negative regulatory factor (NRF) which repressed transcription from the 3.9 kb start site (Rajput, et al., 1996). A model of transcriptional regulation proposed that the housekeeping promoter was utilized in the expression of the 4.1 kb transcript, whilst transcription from the 3.9 kb start site was controlled by an adjacent tissue-specific promoter. In somatic cells expressing low levels of |3l,4-GalTase such as brain tissue, the neuroblastoma cell line N18TG2 and spermatogonia (Shaper, et al., 1990) only the 4.1 kb transcript was synthesized (Harduin, et al., 1993) as NRF would be bound (no 3.9 kb mRNA produced) and only low levels of Spl transcription factor available to bind to the GC boxes upstream from the 4.1 kb start site. Those cells of intermediate pl,4-GalTase expression which included almost all somatic cells, would have more Spl present and a less stable interaction of the NRF resulting in about 5 times more of the 4.1 kb- than the 3.9 kb- mRNA. The mammary glands from mid to late pregnant mice and from lactating mice, which have very high expression of pl,4-GalTase, showed a much greater level of the 3.9 kb- compared to the 4.1 kb-mRNA (10:1) (Harduin, et al., 1993). These tissues would not have any interaction of the NRF, lots of AP2 and a mammary gland specific form of CTF/NFl bound between the 4.1 kb and 3.9 kb start sites and similar amounts of Spl as in the cells expressing intermediate levels of pl,4-GalTase (Rajput, et al., 1996). In addition, the predicted secondary structure in the 5' untranslated region of the 4.1 kb mRNA transcript is extensive and stable, possibly resulting in poor translation compared to the 3.9 kb mRNA (Kozak, 1991; Harduin^, et al., 1993). Elements for such regulation also occur in the 5' end of the bovine (Masibay and Qasba, 1989) and human (Masri, et al., 1988) pi,4-GalTase genes.

pi,4-GalTase is thought of as a housekeeping gene as it is present in most eukaryotic cells and expressed constitutively at low levels. Contrary to this proposed role are studies looking at pl,4-GalTase expression in embryonic development and cell cycle. The expression of pl,4-GalTase was shown to vary in murine embryogenesis with a peak apparent in 13 day old post-coitus embryos and low levels found at days 16 and 19. Re-expression of p 1,4-GalTase was apparent in the adult mouse tissues (Pouncey et al., 1991). The developmental regulation of pi,4-GalTase mRNA was very similar to that of p58GTA mRNA, which expresses a kinase that can activate pl,4-GalTase (Pouncey, et al., 1991). Recent studies on pi,4-GalTase knock-out mice demonstrated that these mice were born normally and were fertile, thus indicating that p 1,4-GalTase expression was not required for fertilization or embryonic development (Asano, et al., 1997; Lu, et al., 1997). These pl,4-GalTase-null mice had stunted growth, pituitary deficiencies, dysregulation of epithelial cell proliferation and differentiation, and high mortality (Asano, et al., 1997; Lu, et al., 1997). p 1,4-GalTase mRNA was upregulated

in chicken embryos during comeal development (Cai et al., 1996). Treatment of mouse F9 teratocarcinoma cells with retinoic acid or retinoic acid plus dibutyryl cyclic AMP causes the cells to differentiate into primitive endoderai and parietal endoderm-like cells respectively with concomitant increases in p 1,4-GalTase expression (Lopez et al., 1989; Kudo and Narimatsu, 1995). The accumulation of p 1,4-GalTase mRNA in the F9 differentiated cells was due to post-transcriptional stabilization rather than transcriptional activation (Kudo and Narimatsu, 1995). Much higher levels of p 1,4- GalTase enzyme activity compared to P 1,4-GalTase mRNA were expressed in the differentiated cells, possibly as a result of post-translational regulation (Kudo and Narimatsu, 1995).

Semm stimulation of quiescent mouse 3T3 fibroblast cells (Masibay et al., 1991) and human HeLa cells (Pouncey, et al., 1991) led to an increase in the p 1,4-GalTase mRNA, activity and protein levels, p 1,4-GalTase gene expression is regulated in a similar way to early response genes during re-entry into the cell cycle from GO. During the cell cycle, P 1,4-GalTase expression peaks at late G l, S and early G2 phases (Pouncey, et al.,

1991). Superinduction of p 1,4-GalTase mRNA was seen in the presence of inhibitors of protein synthesis (Masibay, et al., 1991; Pouncey, et al., 1991). The time course of P 1,4-GalTase mRNA expression did not parallel that of p 1,4-GalTase protein (Masibay, et al., 1991). Induction of the message peaked at 2-4 hours (early-gene) post-serum stimulation then declined, whereas the level of P 1,4-GalTase protein increased steadily, reaching a maximum at 8 hours and remained stable until the cell divided (Masibay, et al., 1991). Further, the 4-fold upregulation in p 1,4-GalTase protein only resulted in a 2- fold increase in p 1,4-GalTase activity (Masibay, et al., 1991). Both the Golgi and cell surface p 1,4-GalTase were increased following serum stimulation. A slightly greater increase occurred in the population at the cell surface, supporting an additional function for this differentially regulated p 1,4-GalTase species (Masibay, et al., 1991; Hinton, et al., 1995). Similar experiments were conducted using RNase protection assays to distinguish between the long and short p 1,4-GalTase transcripts, indicating that the long transcript was expressed in a biphasic pattern with peaks at 0.75 hours and at 4 hours post-stimulation whilst the short transcript only peaked at 4 hours (Hinton, et al., 1995). Further, an increase in cell surface p 1,4-GalTase expression on mouse 3T3 fibroblasts, correlated with an increased number of cells arrested in the Gl phase of cell cycle (Hinton, et al., 1995). The above studies correlating surface p 1,4-GalTase expression with cell growth would argue that p 1,4-GalTase is a cell cycle dependent gene.

The extent of galactosylation in vivo will not only depend upon the P 1,4-GalTase expression levels, but also its substrate requirements and activities of glycosyltransferases competing for the same acceptor substrate such as the bisecting GnT III (Fujii, et al., 1990; Nishiura et al., 1990). NIH 3T3 fibroblasts stably

transfected with N-ra^-proto-oncogene were induced with dexamethasone to express the ras gene (Easton et al., 1991). These cells were then able to increase the activities of several glycosyltransferases including the bisecting GnT III and branching GnT V and the elongating enzymes p 1,4-GalTase and p i,3 GnT resulting in higher molecular weight N-linked glycans at the cell surface (Easton, et al., 1991). Although dexamethasone can induce ST6Gal I activity via increased transcription (Wang et al., 1989), the effects seen in these transfected cells were not due to the action of dexamethasone alone (Easton, et al., 1991).

Poor correlation between in vivo (human tissues) and in vitro (transformed cell line) particular glycosyltransferase mRNA levels have been found (Li et al., 1995). Sometimes changes in glycosylation may result in little effect in vitro yet have profound consequences in vivo (Varki, 1993). For example cells lacking GnT I, which is a key enzyme in converting high mannose N-linked oligosaccharides into complex and hybrid chains, are viable (Stanley et al., 1975). However, a lack of GnT I activity in vivo results in embryonic death by day 10.5 (Ioffe and Stanley, 1994; Metzler et al., 1994).

Obviously the subcellular localization of p 1,4-GalTase will influence its functional role. It has been proposed that Golgi and plasma membrane p 1,4-GalTase are two independently regulated pools of cellular P 1,4-GalTase (Eckstein and Shur, 1989; Lopez, et al., 1991; Masibay, et al., 1991; Evans, et al., 1995). If the long form of p 1,4- GalTase does truly locate preferentially to the plasma membrane (see section 1.2.4) then transcriptional control could alter its subcellular location. Different levels of p 1,4- GalTase expression have been found in various tissues as has the differential use of promoters (see above).

1.3.6.2 Post-translational regulation of pi,4-GalTase activity

Post-translational modifications of p 1,4-GalTase include glycosylation, sulphation, palmitation, and phosphorylation (Strous, 1986). The oligosaccharide moieties on p 1,4- GalTase were shown to have no effect on the enzymatic activity (see section 1.2.3). Sulphation has not been studied in relation to p 1,4-GalTase activity but contributes to the charge heterogeneity (Strous, 1986). Palmitation was shown to occur on the two precursor and mature P 1,4-GalTase polypeptides (Strous, 1986). Although the role of palmitation on p 1,4-GalTase has received little interest, one report indicated that palmitate and also phosphatidylcholine could upregulate human kidney p 1,4-GalTase activity, involved in the synthesis of lactosylceramide (Chatterjee and Ghosh, 1990). Similar effects were also observed on the soluble bovine milk p 1,4-GalTase, whilst other acid lipids, such as phosphatidylserine and phosphatidic acids, inhibited the activity (Mitranic et al., 1983). A much shorter fatty acid chain, n-butyrate, a known

inducer of gene expression {Kruh, 1982), has been shown to have variable effects on glycosyltransferases, causing a 2.5-fold increase in p 1,4-GalTase activities (Li, et al.,

1995).

Phosphorylated serine residues in p 1,4-GalTase has been described which contribute to the charge heterogeneities seen in p 1,4-GalTase (Strous, et al., 1987) and resulted in higher enzymatic activities (Bunnell et al., 1990a), The possibility of O-linked sugars being phosphorylated was not discounted (Strous, et al., 1987). The phosphorylated forms of p 1,4-GalTase were detected primarily in Golgi-associated p 1,4-GalTase in HeLa and HepG2 cells, with very little found in secreted soluble p 1,4-GalTase, suggesting that the negatively charged groups may be involved in cell retention of the P 1,4-GalTase (Strous, et al., 1987). Humphreys-Beher et al (1986) isolated a cDNA clone from a human expression system using an anti-p 1,4-GalTase antibody. This clone was thought (wrongly) to code for human p 1,4-GalTase. Further studies showed that this clone encoded a calmodulin-regulated serine/ threonine protein kinase, p58^TA^ which showed a high degree of sequence homology with the p34cdc2 kinases, a family of cell division control kinases. (Bunnell et al., 1990c; Kidd et al., 1991). The p58^TA is also involved in the cell cycle of CHO cells and in apoptosis (Bunnell, et al., 1990c; Lahti et al., 1995). High level expression of p58^TA COS cells retarded cells in the early Gl phase of cell cycle (Bunnell, et al., 1990c) and caused a concomitant increase in the p 1,4-GalTase activity post-translationally, but no effect on ST6 activity was observed (Bunnell, et al., 1990a). Conversely, a decreased level of p58^TA kinase expression in CHO cells correlated with increased DNA synthesis and with low p 1,4- GalTase activity (Bunnell et al., 1990b). However in one study, no correlation was found between p 1,4-GalTase activity and what is now known to be the p58^TA cDNA, in particular no mRNA p58^TA transcript was detected from mouse lactating mammary gland, a very rich source of p 1,4-GalTase expression (Lopez and Shur, 1988). Nonetheless, p58^TA was able to phosphorylate p 1,4-GalTase in 'vitro with an approximately three-fold increase in activity, whereas incubation in the presence of phosphatase resulted in about a six-fold decrease in activity (Bunnell, et al., 1990a). Another calmodulin-dependent protein kinase isolated from isoproterenol-stimulated proliferating rat parotid acinar cells was also able to phosphorylate serine residues on P 1,4-GalTase, but this had no effect on p 1,4-GalTase activity (Purushotham et al., 1992). P 1,4-GalTase contains an SPHK phosphorylation consensus sequence used by this cdc2 kinase family which is located within the soluble portion of P 1,4-GalTase (Figure 1.1). This site could be phosphorylated when p 1,4-GalTase is located within the Golgi, possibly even involved in retention, and dephosphorylated on the secreted P 1,4-GalTase as the cleaved enzyme does not contain much phosphorylation (Strous, et al., 1987).

It would be interesting to see if P 1,4-GalTase contained any O-GlcNAc which could be potential reciprocal phosphorylation sites as a means of regulating protein activity and/ or function (Hart, et al., 1995). Soluble bovine milk p 1,4-GalTase does not contain any O-GlcNAc (Doris Snow, University of Alabama, USA, personal communication), but as the majority of these post-translational modifications occur in the cytoplasm, the cytoplasmic tail of p 1,4-GalTase which contains two serines on the long form and one serine on the short form, may be modified.