Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Received on May 28, 1999; revised on July 14, 1999; accepted on July 21, 1999.
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Abstract |
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Key words: sialyltransferase/gene structure/B cell differentiation/multiple mRNA
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Introduction |
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The attachment of 2,6-linked sialic acids to Galß1,4GlcNAc-R termini is mediated by the ß-galactoside
2,6-sialyltransferase, ST6Gal I (SiaT1, ST6N, EC2.4.99.1). Mice unable to express ST6Gal I exhibit severe B cell deficiencies, including absence of T-dependent and T-independent responses, reduced circulating IgM levels, and impaired proliferative response to IgM and CD40 crosslinking (Hennet et al., 1998
). The more drastic consequence of ST6Gal I ablation compared to CD22 mutation strongly suggests, in addition to extracellular signaling with CD22, other roles for
2,6-sialyl linkages in B-lineage cells.
Maturation and activation of B cells are accompanied by elevation of ST6Gal I expression (Bast et al., 1992; Keppler et al., 1992
; Aasheim et al., 1993
). Human lymphoblastoid cell lines, exhibiting the mature, active B-phenotype, express a class of ST6Gal I mRNA that retains the complete ST6Gal I protein coding domain and differs from other ST6Gal I mRNA isoforms only in the 5'-untranslated region (Wang et al., 1993
). Transcription of this class of ST6Gal I mRNA is regulated by P2, a B cell specific promoter previously described in the human gene (Lo and Lau, 1996
, 1999). Expression of ST6Gal I is not limited to B-lineage cells. In liver, ST6Gal I participates in the acute phase response (Kaplan et al., 1983
; Jamieson et al., 1987
). Human hepatic ST6Gal I gene expression is mediated by P1, a liver-specific promoter that is physically distinct from P2 (Lo and Lau, 1996
). A third distinct promoter, P3, mediates ST6Gal I gene expression in other cell types, including pre-B cells. Transcripts originating from P3 incorporates sequences from human Exons Y and Z (Wang et al., 1993
) or the corresponding Exons 1 and 0 in rat (Wen et al., 1992
).
Earlier, we have identified the mouse homologue of the P1 promoter and its associated hepatic transcript from the mouse ST6Gal I gene, Siat1 (Hu et al., 1997). Here, we extend this analysis to Siat1 expression in mouse B cells. We report that four distinct ST6Gal I mRNA forms are associated with the transition from resting B cells to plasma cells. One of these is the major Siat1 mRNA isoform in resting B cells and is likely the mouse homologue to the previously reported human Exons Y+Z and rat Exons 1+0-containing forms. Three other mRNA forms, whose expression are likely restrict to B-lineage cells, are induced during B cell activation and differentiation. Each of these mRNA forms is transcriptionally initiated at physically well-separated points, and by inference, under the control of physically distinct promoter regulatory regions. The separate regulatory programs govern that the expression of each of these mRNA forms is under separate programs of expression. The exons encoding the various 5'-UT regions are dispersed in over 69 kb of linear genomic sequence.
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Results |
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PCR amplified signal from the X3-specific primer generated a complex series of products with the most abundant product located at 1 kb (Figure 3, lane d). However, none of the ethidium bromide visible bands hybridized positively for X3, suggesting that these products are artifacts of the RT-PCR procedure. Sequence analysis of cloned products corresponding to the 1 kb, 550 bp, and 430 bp regions confirmed that none contain recognizable segments of the neither X3 sequence nor genomic regions flanking the X3 exon. However, an X3-positive signal was detected at 100 bp region that was barely noticeable by ethidium bromide staining (data not shown). Nine clones were generated and sequenced from the 100 bp area, and their sequence confirms the authenticity of X3. There were no X1, X2, or O sequence regions in these nine clones. The largest cloned product predicts a 73 nt X3 region.
To characterize further the divergent mRNA forms containing X1, X2, X3, and Q+O, mRNA from day 3 was used to program RT-PCR using, as upstream primers, oligonucleotides corresponding to the divergent regions X1, X2, X3, and Q+O. The downstream primer is complementary to the region encompassing the translation termination codon in Exon VI. The PCR-amplified signals, as shown in Figure 4 by ethidium bromide staining, are the predicted sizes consistent with the inclusion of the complete ST6Gal I ORF in each of these mRNA isoforms.
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Discussion |
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Genes of higher vertebrates are often conveniently classified as either housekeeping or tissue-specific genes. ST6Gal I, GalT1, and many other glycosyltransferases, however, exhibit characteristics of both categories. GalT-1, a ß1,4-galactosyltransferase, is normally transcribed in a constitutive mode in all cells (Rajput et al., 1996). However, in lactating mammary epithelium and in testes, additional transcription regulatory regions participate to generate distinct GalT-1 mRNA isoforms that enable high level expression. ST6Gal I is expressed in most tissues and cells, a property consistent with a housekeeping gene (Paulson et al., 1989
; Wen et al., 1992
). On the other hand, ST6Gal I expression is particularly high in certain tissues, a characteristic of tissue-specific genes (Svensson et al., 1990
, 1992; Wang et al., 1990a
). In liver, ST6Gal I participates in the hepatic acute phase reaction and sialylation of serum glycoproteins (Kaplan et al., 1983
). Regulated expression of ST6Gal I in liver is under the control of P1, a hepatic-specific promoter that is responsive to glucocorticoids and IL-6 (Wang et al., 1990b
; Dalziel et al., 1999
).
We previously reported a human ST6Gal I mRNA form expressed in B lymphoblastoid cells (Wang et al., 1993). The human B cell mRNA contains the unique 5'-UT exon, Exon X. At present, it is not known which among the mouse Exons X1, X2, or X3 is the murine homologue to the human Exon X. No significant sequence similarity exists between human Exon X and the mouse sequences reported here. In the earlier report, we documented that resting and immature human B cells express an Exons Y and Z-containing form. We reported that the Exon Y+Z-containing form is likely the human homologue to the rat constitutively expressed Exons 1+0 form (Wen et al., 1992
), since substantial sequence similarity is shared between rat Exon 0 and human Exon Z (Wang et al., 1993
). The mouse Exons Q+O form reported here is likely the mouse homologue of the constitutively expressed transcript; significant sequence similarity is shared between mouse Exon O and human Exon Z (68%) or mouse Exon O and rat Exon 0 (91%).
Our data strongly implicate unique and well-separated transcription initiation points for each of the mouse ST6Gal I transcripts expressed in B-lineage cells. The strongest evidence is the mutually exclusive usage of the 5'-UT exons. Exons Q and O-containing form have been previously characterized as the constitutively expressed form in most cells and tissues. Among the known 5'-UT exons, Exons Q and O are also the most 5' distal exons from the coding exons (see Figure 5). RT-PCR using a primer pair against sequences in Exon O and Exon VI resulted in the predicted sized product consistent with the inclusion of the complete ST6Gal I coding sequence. However, there is no evidence for inclusion of the intervening 5'-UT exons, as indicated by the absence of size polymorphism in the Exons O to VI product (see Figure 4). Furthermore, 16 independent clones of Exon O-containing 5'RACE products from B cells have been analyzed to date. None incorporate any of the 5'-UT exons (i.e., as X2, H, X1, or X3) that lie between Exon O and Exon I. The sum of these observations strongly implicate that transcriptional initiation site utilized to generate the Exon O-containing mRNA does not generate the precursors for mRNA isoforms containing X2, H, X1, or X3. Likewise, transcripts containing X2 as the 5'-most exon do not contain the more proximal 5'-UT exons, H, X1, or X3, as evidenced by 5'-RACE and RT-PCR data. This implies that transcription initiation events giving rise to X2-containing transcripts do not generate H, X1, or X3-containing forms. The same rationale and observations apply for H, X1, and X3-containing mRNA forms.
Two principal lines of evidence strongly indicate that the complete ST6Gal I coding information is present within each of the isoforms. First, the sizes of the RT-PCR products are fully consistent in the presence of the complete ORF within forms containing X1, X2, X3, and Q+O (see Figure 4). Second, probes against these divergent regions hybridize only to the same sized region on Northern blots (see Figures 2 and 7), suggesting the absence of heterogeneous or truncated transcripts. The transition from resting B cells to plasma cells is a multistep process involving cell activation, apoptotic selection, and clonal expansion. In additional to plasma cells, memory B cells, a class of cells that is not yet well understood, also arises from B cell activation and differentiation. We have shown here that a number of distinct ST6Gal I mRNA isoforms is expressed in the activation and differentiation of resting splenic B cells. These isoforms, putatively under the control of separate transcription regulatory regions, follow unique time courses of expression.
However, at present it is also not known if each isoform is restricted to a unique subset of the represented B cell population, where they may be utilized in sialylation for functionally divergent reasons. Clearly, ST6Gal I expression during the early stages of B activation coincides with the ability to synthesize ligand for CD22. As mentioned earlier, mice unable to elaborate ST6Gal I exhibit more striking B cell deficiencies than the CD22-null animals. The severity of these defects strongly suggests additional requirements of appropriate 2,6-sialylation in B cell function. Consistent with this view is the presence of ST6Gal I mRNA isoforms that are expressed during the B cell development pathways and are under independent programs of regulation.
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Materials and methods |
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Recombinant CD40-ligand (CD40L) was also a generous gift from Marilyn Kehry. Sf9 insect cells expressing CD40-ligand were used to prepare cell membrane extracts as described previously (Hodgkin et al., 1994) and kept frozen at 70°C until use.
Preparation of B cells
Male and female C57BL6 wt mice were obtained from Taconic Laboratories (Germantown, NY). Typically, 1530 animals of mixed sexes between 8 and 12 weeks of age were used for each preparation of resting B-splenocytes as described previously (Hodgkin et al., 1990, 1994). Briefly, a single cell suspension was prepared by teasing spleens from freshly sacrificed mice through a stainless steel mesh. The cell suspension was depleted of RBC using hypotonic ammonium chloride solution, and adherent cells were depleted by incubation in plastic tissue culture dishes for 1h. T cells were depleted by complement lysis using first anti-Thy1.2 (30H12) and anti-CD4 (GK-1.5) followed by incubation with Low-Tox rabbit complement (Accurate). B cells were further purified by centrifugation on a Percoll density gradient; resting B cells were recovered from the 65/72% Percoll gradient interface. Cells were 60% CD19+ and <4% CD3e+ as determined by flow cytometry.
In petri dishes, B cells were cultured in B cell medium (BCM: RPMI 1640 supplemented with 10% FBS (Hyclone, defined), 10 mM HEPES (pH 7.3), 2 mM L-glutamine,1 mM nonessential amino acids, 1 mM sodium pyruvate, 5 x 105 M 2-mercaptoethanol, and 50 µg/ml genamicin sulfate). To induce in vitro activation and differentiation, D10 supernatant and CD40L was added to cultures at 1/100 and 1/800, respectively. Cell cultures were split 1:2 with fresh medium containing D10 supernatant and CD40L (see Reagents above) on days 3, 5, and 7 of culture.
In vitro determination of B cell differentiation
Secretion of Ig was determined using Western blot analysis. Briefly, cells were harvested and the supernatant saved. Using the Bio-Rad Mini gel apparatus, 10 mg of supernatant for each time point was loaded on three 12% acrylamide resolving gels. Proteins were transferred to Zetabind PVDF membranes (Cuno). The blots were probed for IgE, IgG, and IgM. IgE was stained in two steps using first rat anti-mouse IgE (Zymed) followed by alkaline phosphatase-goat anti-rat IgG (H+L) (Zymed). IgG and IgM were stained in a single step with either alkaline phosphatase goat anti-mouse IgG, Fc Fragment specific or alkaline phosphatase goat anti-mouse IgM, µ chain specific antibodies obtained from Jackson ImmunoResearch Laboratories. Alkaline phosphatase was detected using CDP-Star Western Blot Chemiluminescence Reagent (DuPont NEN).
CD22 mRNA levels were determined by RT-PCR. cDNA was generated by random hexamer-primed reverse transcription from 2 µg of total RNA. The resultant cDNA was subjected to PCR using CD22-specific primers, CD22(s) and CD22(as). PCR parameters for the Perkin-Elmer 2200 thermocycler are 95°C for 1 min, 62°C for 90 sec, 72°C for 90 sec, 28 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 90 sec, then 72°C for 7 min and 4°C. CD22 products are visualized on 3% agarose gels.
RNA and RT-PCR analysis
Total RNA was prepared from cultured B cells for each time point using guanidine isothiocyanate method (Chirgwin et al., 1979). RNA samples were checked for integrity on agarose gels by probing for RPL (ribosomal protein RPL32-27.3.7). Small differences in RNA concentration among samples were normalized by RPL signaling.
For 5'-RACE, the Marathon cDNA Amplification kit from Clontech was used. Twenty µo
µ
of total or 1 µg of poly(A)+ RNA was annealed to the primer mST1-PII and reverse transcribed. mST1-PII is complementary to a region in Exon II, such that an authentic reverse transcription events of ST6Gal I mRNA must span at least the Exon IExon II boundary. The cDNA product was ligated to the anchor primer as per instructions and subjected to PCR using the anchor primer AP1 and unless otherwise stated, the ST6Gal I Exon I-complementary primer mST1-P4. PCR parameters using Perkin-Elmer GeneAmp System 2400 were 94°C for 2 min, 63°C for 2 min, 72°C for 2 min, 35 cycles of 94°C for 45 sec, 63°C for 45 sec, 72°C for 2 min, then 72°C for 7 min and hold at 4°C. PCR products were then visualized on 1.5% agarose gels. Where appropriate, PCR products were also cloned into a plasmid vector (pCRII from Invitrogen or pBluescript SK+ from Clontech) and sequenced.
Using cDNA generated as described, real-time PCR was performed as per instructions for using the TaqMan Universal PCR Master Mix supplied by Perkin-Elmer: Applied Biosystems. Primers and probes were designed using Primer Design software (Perkin-Elmer: Applied Biosystems). A 32 nt oligo Taqman probe ST1EI (5'-aaa gta aac ctc ttt ccc gtg gag aac agt gC-3') was designed in the Exon I region of ST6Gal I. The probe was covalently linked with the reporter dye FAM at the 5'end and the quencher dye TAMRA at the 3' end (and synthesized by ABI). A common reverse primer, mST1-p332 to each of the isoforms was designed in Exon I downstream of the probe. Unique forward primers to each isoform were designed in the upstream exons of interest: X1 (mST1-p333), X2 (mST1-p336), X3 (mST1-p377), and O (mST1-p335). As an internal control, a probe (5'-CCA ACG CCA GGT ACG CAG CGA A-3') labeled with the reporter dye JOE and TAMRA and primers, RPL-p338 and RPL-p339 were designed for the ribosomal protein L32. For ST6Gal I reactions, the final reaction mixture of 50 µl consisted of 300 nM of mST1-p332, 50 nM of forward primer, 150 nM of ST1EI probe and 25 µl of 2x Taqman Universal PCR Master Mix. PCR parameters for ABI Prism 7700 are 50°C for 2 min, 95°C for 10 min, 64 cycles of 95°C for 15 sec, and 62°C for 1 min.
Isolation and analysis of mouse genomic DNA
Mouse lambda genomic library (Hu et al., 1997) was screened using exon-specific sequences as probes. Clones containing Exons I, II, X1a, X1b, X2, X3, Q, O, and H were obtained and characterized. In order to determine the relative locations of the exons, two overlapping BAC clones, were obtained by screening a BAC library. Long distance PCR (Siebert et al., 1995
) was also used to determine the distance between Exon H and Exon X1 using the Advantage Genomic Polymerase Mix (Clontech).
Summary of oligonucleotide primers
Synthetic oligonucleotides used are: CD22(s) 5'-ACC TCC ATC ACC TCT TCT GT-3'; CD22(as) 5'-GTT CCA CTT CTT CCG ACT CT- 3'; mST1-PII 5'-GCA GAT GAT GGC AAA CAG GAG-3'; AP1 5'-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3'; mST1-P4 5'-TCT GGC TAA TCC TTC TGG GC-3'; mST1-p332 5'- CTC CAG GTC CTC AGG AGC C-3'; mST1-p333 5'-TCC TTT CCA TCA CTG TCT GCC T-3'; mST1-p336 5'-CCG GCA AGG TCC ACT TAC AAT-3'; mST1-p377 5'-AAG CTT CAG AAG AGT GGT TAA TGG TT3'; mST1-p335 5'-AGC CGG ATG CTG AAT GGT T-3'; RPL-p338 5'-CAT GCA CAC AAG CCA TCT ACT CA-3'; RPL-p339 5'-TGC TCA CAA TGT GTC CTC TAA GAA C-3'; mst1ex6(as) 5'GGA GAG GAG GAT GTT GTC AG-3'; X1P1(as) 5'-GGA GTC CCA CAC CAA ACA CCA ATC TGC TGC ATT-3'; X2P1 5'-AAG TGG ACC TTG CCG GCA CA-3'; F249 5'-CCA GGC AGA CAG TGA TGG AAA GGA T-3'; X3P1 5-ACC ACT CTT CTG AAG CTT TA-3';mST1-p203 5'-ttg gtg ttt ggt gtg gac tc-3'; mST1-p304 5'-AGT GGA AGG AAG CTA GGA GGG-3'; mST1-p206 5'-TGA GCC TTC CCC AAA TAC CTG-3'.
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Acknowledgments |
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Footnotes |
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2 Present address: Piscataway, NJ
3 To whom correspondence should be addressed
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References |
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