Murine B cell differentiation is accompanied by programmed expression of multiple novel ß-galactoside {alpha}2,6-sialyltransferase mRNA forms

Sherry A. Wuensch, Ruea Yea Huang, Jonathan Ewing1, XueLian Liang2 and Joseph T. Y. Lau3

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
ST6Gal I (ß-galactoside {alpha}2,6-sialyltransferase, ST6N) elaborates the ubiquitously expressed {alpha}2,6-sialyl linkage. A number of ST6Gal I mRNA isoforms, differing only in their 5'-UT regions, is transcribed from a single mouse gene, Siat1. In B-lineage cells, {alpha}2,6-sialic acid serves as extracellular ligand for CD22, a participant in cell activation via an intracellular signaling network of tyrosine kinases and SHP phosphastase. Activation and terminal differentiation of mature B cells into plasma cells is accompanied by the appearance of at least four distinct ST6Gal I mRNA isoforms. Resting splenic B-lymphocytes isolated from 8–12 wk C56Bl/6 mice expressed almost exclusively the Exons Q+O-containing form, which is the likely homolog to the previously documented human Y+Z and rat –1+0 forms. In vitro activation using recombinant CD40-ligand and conditioned media from T-helper cells resulted in a 2- to 3-fold elevation of overall ST6Gal I mRNA abundance by Day 3. This coincided with repression of the Q+O form, and appearance of three new isoforms containing 5'-untranslated sequences X1, X2, or X3. The X1 form persisted through Day 10, when the transition of B cells to plasma cells was completed as evidence by disappearance of CD22 mRNA. In contrast, the X2 form only transiently appeared at Day 3 and declined to barely detectable levels by Day 7. Expression of the X3 form, a minor mRNA form, paralleled the X2 form. The divergent 5'-UT exons are dispersed over 69 kb of linear genomic space of Siat1. Mutually exclusive utilization of these 5'-UT exons in transcripts predicts separate and distinct promoter regulatory regions for each mRNA isoform.

Key words: sialyltransferase/gene structure/B cell differentiation/multiple mRNA


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
B cell activation and differentiation are accompanied by dramatic reorganization of cell surface carbohydrates, as exemplified by the zones of peanut agglutinin (PNA) reactivity that have long served as structural reference points within germinal centers. Among B cell surface components that participate in B cell activation, CD22 is an accessory molecule thought to negatively modulate the BCR (B cell receptor complex) via intra­cellular signaling networks of SHP1 protein tyrosine phosphatase and protein tyrosine kinases (Cyster and Goodnow, 1997Go; O'Rourke et al., 1997Go; Tedder et al., 1997Go). Absence of CD22 results in tyrosine phosphorylation of cellular proteins occurring at a lower level and an increase in intracellular calcium following BCR crosslinking (O'Keefe et al., 1996Go; Sato et al., 1996Go; Nadler et al., 1997Go; Nitschke et al., 1997Go). The extracellular domain of CD22 has a lectin-like component that specifically recognizes {alpha}2,6-linked sialic acids (Law et al., 1995Go; vander Merwe et al., 1996Go; Razi and Varki, 1998Go).

The attachment of {alpha}2,6-linked sialic acids to Galß1,4GlcNAc-R termini is mediated by the ß-galactoside {alpha}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., 1998Go). The more drastic consequence of ST6Gal I ablation compared to CD22 mutation strongly suggests, in addition to extracellular signaling with CD22, other roles for {alpha}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., 1992Go; Keppler et al., 1992Go; Aasheim et al., 1993Go). 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., 1993Go). 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, 1996Go, 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., 1983Go; Jamieson et al., 1987Go). Human hepatic ST6Gal I gene expression is mediated by P1, a liver-specific promoter that is physically distinct from P2 (Lo and Lau, 1996Go). 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., 1993Go) or the corresponding Exons –1 and 0 in rat (Wen et al., 1992Go).

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., 1997Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
ST6Gal I expression during in vitro differentiation of resting splenic B cells into plasma cells
Resting splenic B lymphocytes in culture, when exposed to conditioned media from T-helper cells and recombinant CD40-ligand (CD40L), activate and terminally differentiate into plasma cells. Accumulation of secreted IgG and IgM was observed within 3–5 days (Figure 1, A and B, respectively). Secreted IgE was detectable in the media by days 5–7 (Figure 1C). Intracellular pool of CD22 mRNA is repressed by day 3 (Figure 1D), which correlates with Ig secretion and was in agreement with documented patterns of expression for CD22 in murine B cells (Erickson et al., 1996Go; Stoddart et al., 1997Go).



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Fig. 1. In vitro activation and differentiation of resting B splenocytes. Western blot analysis for secreted IgG, IgM, and IgE (A, B, and C, respectively) was performed on resting B cells after stimulation with D10 and CD40L as described in Materials and methods. Media supernatant was collected during day 0 to 3 (lane 3), day 3 to 5 (lane 5), day 5 to 7 (lane 7), and day 7 to 10 (lane 10). D10 and CD40L were added at Day 0. Media prior to exposure to cells were also analyzed (lane M). In (D), CD22 mRNA was assessed by RT-PCR from cells cultivated for 0, 3, 7, or 10 days (lanes 0, 3, 7, and 10, respectively). Lane b is a parallel RT-PCR reaction programmed without input RNA.

 
ST6Gal I mRNA, as probed on Northern blot for Exon II, is elevated 3- to 4-fold by day 3 (Figure 2, open bars), corresponding with activation of the resting B cells (Hodgkin et al., 1994Go). Exon II encodes the translational initiation codon and is invariant in all known forms of ST6Gal I mRNA. Earlier, we identified a ST6Gal I mRNA isoform from spleen RNA of young adult 129/J mice. This mRNA isoform contains the intact ST6Gal I protein coding domain and differs only in a unique 5'-most untranslated region, termed X1 here (Liang and Lau, unpublished observations). This mouse isoform was presumed to be the murine equivalent of the previously documented human Exon X–containing isoform (Wang et al., 1993Go). Activation and differentiation of resting splenic B cells also resulted in induction of the X1 sequence (Figure 2, shaded bars).



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Fig. 2. ST6Gal I mRNA is elevated upon in vitro activation of resting B splenocytes. Northern blot of RNA from cells cultivated for 0, 3, 7, or 10 days in the presence of CD40L and D10 was probed for ST6Gal I Exon II or for ST6Gal I Exon X1 (inset, A and B, respectively). The hybridization signals were quantified by phosphorimager scanning and normalized to the ribosomal protein mRNA control RPL (see Materials and methods). The RPL-normalized Exon II (open bars) and Exon X1 (shaded bars) signals are summarized in graph format.

 
Novel ST6Gal I mRNA isoforms
5'-RACE analysis was performed for a qualitative assessment of the ST6Gal I mRNA isoforms. RNA was primed for reverse transcription with an oligonucleotide complementary to a region in Exon II. Presence of Exon I sequence in the cloned 5'-RACE products served as criteria for clones authentically derived from ST6Gal I mRNA. Four definitive types of Exon I–containing clones, X1, X2, X3, and Q+O, differing only in the region 5' of the Exon I sequence were generated. This is summarized in Table I, where the unassigned category represents Exon I–containing clones that failed to extend beyond Exon I. X1 is the divergent sequence previously identified from mouse spleen ST6Gal I mRNA (Liang and Lau, unpublished observations). The Q+O species is probably equivalent to the previously identified human Y+Z (Wang et al., 1993Go) and rat Exon –1+0 (Wen et al., 1992Go). The mouse Exon O is 68% and 91% identical to human Exon Z and rat Exon 0, respectively (data not shown). X2 and X3 are novel sequences not previously observed.


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Table I. ST6Gal I mRNA isoforms expressed by in vitro differentiating B cellsa
 
In order to determine the relationship and origin among the isoforms, mRNA from day 3 was subjected to a modification of the 5'-RACE analysis as described in the previous paragraph, where the 3' PCR primers were sequences complementary to X1, X2, or X3 regions. When downstream primers complementary to X1 were used, products of 90 ± 20 bp and 190 ± 20 bp were generated, based on ethidium bromide staining of agarose gels (Figure 3, lanes a and b, respectively). The size difference was due to the locations of the two X1-specific primers. More importantly, when the 5'-RACE product was subjected to Southern analysis, there was no hybridization to probes for X2, X3, or O (data not shown). Thirteen individual 5'-RACE clones were sequence analyzed, with the largest specifying a 267 nt unique 5' sequence for X1.



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Fig. 3. 5'-RACE analysis of the ST6Gal I mRNA isoforms. 5'-RACE analysis was performed on RNA from cells cultivated in the presence of CD40L and D10 for 3 days. One µg of mRNA was subjected to reverse transcription using the Exon II-specific primer mST1-PII (see Materials and methods). PCR amplification was performed using the anchor primer and an exon-specific primer as primer pairs. The exon-specific primers are: X1-specific, X1P1 (lane a); X1-specific, F249 (lane b); X2-specific, X2P1 (lane c); and X3-specific, X3P1 (lane d). Molecular weight markers are also shown (lane M). The PCR conditions were the same as previously described for 5'-RACE (see Materials and methods). Shown is the ethidium bromide visualization of the RACE products after electrophoretic separation on 3% agarose gels.

 
When downstream primers complementary to X2 were used for the PCR, three diffused bands of 140 ± 10 bp, 120 ± 10 bp, and 75 ± 10 bp by ethidium bromide staining were observed (Figure 3, lane c). Sequence analysis indicates the smaller sized products are 5'-truncations of the 140 bp product, putatively arising from premature termination of the reverse transcriptase. Again, when the 5'-RACE product was subjected to Southern analysis, there was no hybridization to probes for X1, X3, or O (data not shown). The largest cloned 5'-RACE product, corresponding to the 140 bp band, predicts a 139 nt unique 5' sequence for X2.

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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Fig. 4. RT-PCR analysis for coding region in the ST6Gal I mRNA isoforms. RT-PCR analysis was performed on RNA from cells cultivated in the presence of CD40L and D10 for 3 days. Two µg of total RNA were used in the initial reverse transcription using the random hexamer nucleotides. For PCR amplification of the cDNAs, mst1ex6(as), a complementary to Exon VI and 3' of the ST6Gal I protein termination codon, and specific primers mST1-p203 (X1), mST1-p336 (X2), mST1-p304 (X3), and mST1-p206 (O) were used. Shown is the ethidium bromide visualization of RT-PCR products after electrophoretic separation on a 1.5% agarose gel.

 
Siat1, the mouse ST6Gal I gene
BAC mouse genomic clones and mouse lambda genomic clones were identified by hybridization to known sequences to Siat1. The 5' structure of the Siat1 (Figure 5) was elucidated by sequence analysis and Southern blotting. Exons encoding the known 5'-UT regions are dispersed over 69 kb of linear genomic sequence. Most proximal to the shared exons is the 73 bp X3 exon, located 5.5 kb 5' of Exon I. The 267 bp X1 sequence is divided into two exons, X1a and X1b, that are interrupted by a 232 bp intron. X1a and X1b reside 3.8 kb 5' of X3. Exon H, the hepatic-specific Exon and transcription start site P1, resides 20 kb 5' of X1. The 139 bp X2 exon is located 18 kb 5' of Exon H. Further upstream is Exon O, ~7 kb 5' of X2, and Exon Q, ~6 to 9 kb 5' of Exon O.



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Fig. 5. Structure and organization of the unique 5'-UT ST6Gal I exons. The known exons of the mouse ST6Gal I gene, Siat 1, is schematically represented. The spatial arrangement and partial restriction map of the 5'-UT exons Q, O, X2, H, X1a, X1b, and X3 are shown to scale, along with putative transcription initiation sites P3, P2b, P1, P2a, and P2c. Roman numerated exons (I to VI) are the shared exons, and the distances between them are not shown to scale. The coding sequence for ST6Gal I protein resides in Exons II–VI. Sequences for the 5'-UT exons, summarized below the schematic, are deposited in GenBank and are assigned accession numbers: AF153680 (Q), AF153681 (O), AF153682 (X2), U67989 (H), AF153684 (X1a and X1b), and AF153683 (X3).

 
Expression of the novel ST6Gal I mRNA Isoforms
Quantitative assessment of ST6Gal I mRNA expression during in vitro B cell development was performed by RT-PCR using real-time fluorescence detection of PCR product (see Materials and methods). As shown in Figure 6, activation and differentiation of splenic B cells result in a linear increase of the X1 isoform with a profile that mirrors that obtained using conventional Northern blot analysis (see Figure 2). Likewise, total ST6Gal I mRNA, as measured by real-time fluorescence-PCR (not shown), was in complete agreement with the data obtained by Northern blots. The Q+O form, as quantified by assessing the level of Exon O-containing transcripts, appears to be the dominant ST6Gal I transcript in resting B cells (day 0). Expression of the Q+O form declined significantly after activation. In contrast, isoforms X2 and X3, are not abundantly expressed until day 3 but decline significantly thereafter. By day 10, the dominant ST6Gal I transcript appears to be the X1 form.



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Fig. 6. ST6Gal I mRNA isoforms are differentially expressed during B cell differentiation. RNA isolated from cells cultivated in the presence of CD40L and D10 for 0, 3, 7, or 10 days was assayed for X1, X2, X3, and Q+O ST6Gal I mRNA isoforms assayed using real-time PCR as described in detail in Materials and methods. Shown are the relative levels of each of the mRNA isoforms during cell activation and differentiation. As in Northern blot analysis using different probes for each isoform, relative levels between the different isoforms are not comparable by this approach.

 
Figure 7 is a multiple mouse tissue Northern blot hybridized for Q+O, X1, X2, X3, and the shared coding region. It should be noted that the ST6Gal I isoforms are visualized using probes each unique not only in sequence but also in length and detection sensitivity. Hence, signal intensities may not accurately reflect the proportion of each of the isoforms within a given sample. Nevertheless, when the level of expression of each isoform are examined among different tissue types, the Q+O form is expressed in all tissue types, consistent with previous observations of corresponding region of the human and rat genes (Wen et al., 1992Go; Wang et al., 1993Go). The Q+O form is also quite evident in day 3 in vitro activated splenic B cells. In contrast, the X1 form was detectable only in spleen among the tissues examined. X2 and X3 forms were below detection limits in RNA prepared from whole tissues, including spleen, although their presence was detectable in day 3 in vitro activated cells.



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Fig. 7. Tissue specificity of ST6Gal I RNA isoforms. Multiple mouse tissue mRNA blots (Clontech Laboratories) were probed for sequences unique to (from top to bottom) Exons Q+O, X2, H, X1, X3, and II. Lanes 1–9 are testis, kidney, muscle, liver, lung, spleen, brain, heart, respectively. RNA isolated from splenic B cells cultivated in the presence of CD40L and D10 for 3 days was also probed for reference (right-most lane). Differential probes recognizing Exons Q+O, X2, H, X1, and II (252 bp, 97 bp, 165 bp, 139 bp, 24 bp, and 750 bp, respectively) were synthesized against PCR-generated fragments of the respective exons sequences and labeled with 32[P]-dATP. The probe against X3 was a 24 nt end-labeled oligonucleotide complementary to X3.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Multiple promoters regulating a common transcribed gene are not an unusual feature in bacteria and lower eukaryotes. An increasing number of vertebrate genes regulated by multiple promoters is also being reported, including, but not limited to, the vitamin D receptor (Crofts et al., 1998Go), the estrogen receptor {alpha} (Flouriot et al., 1998Go), the gonadotropin-releasing hormone (Dong et al., 1997Go), fibroblast growth factor 1 (Chotani et al., 1995Go), the testicular inhibin/activin ßB subunit (Feng et al., 1995Go), and the HMG-I/Y (Ogram and Reeves, 1995Go). Among glycosyltransferases, multiple promoters have been reported not only for ST6Gal I, but also for the {alpha}2,3-sialyltransferase ST3Gal IV (Kitagawa et al., 1996Go), the GlcNAc transferase III (Koyama et al., 1996Go) the GlcNAc transferase V (Saito et al., 1995Go), and a ß1,4-galactosyltransferase (Shaper et al., 1995Go; Rajput et al., 1996Go).

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., 1996Go). 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., 1989Go; Wen et al., 1992Go). On the other hand, ST6Gal I expression is particularly high in certain tissues, a characteristic of tissue-specific genes (Svensson et al., 1990Go, 1992; Wang et al., 1990aGo). In liver, ST6Gal I participates in the hepatic acute phase reaction and sialylation of serum glycoproteins (Kaplan et al., 1983Go). 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., 1990bGo; Dalziel et al., 1999Go).

We previously reported a human ST6Gal I mRNA form expressed in B lymphoblastoid cells (Wang et al., 1993Go). 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., 1992Go), since substantial sequence similarity is shared between rat Exon 0 and human Exon Z (Wang et al., 1993Go). 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 {alpha}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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Reagents
Hybridomas 30H12 (anti-Thy 1.2 producing) and GK1.5 (anti-CD4 producing) were obtained from ATTC and grown as directed. Before use in experiments, culture supernatants were titrated and used for T cell depletion as described (Hodgkin et al., 1994Go). D10 supernatant, containing helper T lymphokines, from activated D10.G4.1 (D10) Th2 cells was a generous gift from Marilyn Kehry (Boehringer Ingelheim). Cytokines present in the D10 supernatant, as assayed by Kehry, were IL-2 (<0.06 ng/mg), IL-3 (414.80 ng/ml), IL-4 (449.88 ng/ml), IL-5 (460.33 ng/ml) IL-6 (781.90 ng/ml), IL-10 (175.27 ng/ml), and GM-CSF (18.66 ng/ml), TNF{alpha} (<1.25 ng/ml), and IFN-{gamma} (<0.3 ng/ml).

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., 1994Go) 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, 15–30 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., 1990Go, 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 10–5 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{gamma} 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., 1979Go). 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 µ{iota}{chi}{rho}o{gamma}{rho}{alpha}µ{sigma} 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 I–Exon 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., 1997Go) 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., 1995Go) 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 TT–3'; 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'.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We thank Dr. Marilyn Kehry for providing the CD40L and D10 and for many helpful discussions. We also thank Dr. Jonathan Lo for his helpful discussions in preparing the manuscript. This work is supported by Grant GM38193 from the National Institutes of Health to J.T.Y.L. and by Institute Core Grant CA16056–21 to Roswell Park Cancer Institute.


    Footnotes
 
1 Present address: Millennium Pharmaceuticals, Inc., 640 Memorial Drive, Cambridge, MA 02139 Back

2 Present address: Piscataway, NJ Back

3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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