tissue-specific expression of ,&galactoside a-2,6-sialyltransferase

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 264, No. 29. Issue of October 15, pp. 17389-17394,1989 Print& in U.S.A.

Tissue-specific Expression of ,&Galactoside a-2,6-Sialyltransferase TRANSCRIPT HETEROGENEITY PREDICTS A DIVERGENT POLYPEPTIDE*

(Received for publication, April 28, 1989)

Terrance P. O’Hanlon, Karen M. Lau, XueCheng Wang, and Joseph T. Y. LauS From the Department of Molecular and Cellular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263

The &galactoside (~-2,6-sialyltransferase represents a member of a family of sialyltransferases which cat- alyze the te rmina l addition of sialic acid to ma tu r ing carbohydrate chains. We surveyed rat tissues using cDNA probes complementary to coding and noncoding domains of the rat liver a-2,6-sialyltransferase. In addition to the expected differences in the level of sialyl- t ransferase mRNA among the tissues, there were dra- mat ic qualitative differences as well. Hepatic sialyl- t ransferase probes hybridize to mRNAs of varying size on Nor the rn blots. A tissue-dependent pattern of expression of these transcripts is documented. Evi- dence is presented that the multiple transcripts are generated f rom a common gene sequence. At least one instance of alternate splicing in the generation of the kidney sialyltransferase transcripts is predicted by S 1 nuclease analysis. We report the isolation of a rat kidney cDNA clone, RKA, that substantiates this tissue- specific alternate splicing event. The RKA insert, although less than full-length, apparently encodes a polypeptide divergent f rom the reported hepatic (u-2,6- sialyltransferase (1). RNA blot analysis indicates that the RKA-type transcripts represent a significant proportion of s ia lyl t ransferase RNA in rat kidney. An- other class of kidney cDNA clones, RKE, is colinear with the hepatic sialyltransferase sequence. RNA blots probed f o r the divergent and common regions suggest that complex processing pa thways are operative in the tissue-specific expression of sialyltransferase mRNA.

The @-galactoside a-2,6-sialyltransferase (EC 2.4.99.1) me- diates the attachment of sialic acid to N-glycosidically linked oligosaccharides that are common to serum glycoproteins ( 2 , 3). In liver, the major site of serum glycoprotein synthesis, the enzyme exists in a predominantly membrane-bound form localized to the Golgi and trans-Golgi network (4) where it participates in the post-translational modification of newly synthesized secretory and cell-surface glycoproteins. The @- galactoside sialyltransferase is translated from a mRNA that includes a 1209-nucleotide open reading frame and an extensive, 3”untranslated region (1). In an architecture that is apparently shared by P-1,4-galactosyltransferase (5-8), the nucleic acid sequence predicts a 46.7-kDa polypeptide com- prised of a short amino-terminal cytosolic region, a single transmembrane domain, followed by an expansive carboxyl-

* This work was supported by Grant GM38193 from The National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Dept. of Molecu-

Buffalo, NY 14263. far and Cellular Biology, Roswell Park Memorial Inst., 666 Elm St.,

terminal catalytic domain. A soluble form of the sialyltransferase in serum is thought to be derived from the liver form (9, 10) by a proteolytic event that liberates the catalytic domain from its membrane anchor (1).

Whereas our current understanding of this sialyltransferase is largely based on studies of the liver system (11-13), it is evident that a-2,g-sialic acid/Gal@l+GlcNAc linkages are not restricted to glycoproteins of hepatic origin. In fact, little is known about the tissue-specific expression of the (r-2,6-sialyltransferase. Here, we report that mRNAs of varying size hybridize with hepatic sialyltransferase sequences and that these multiple forms are expressed in a tissue-specific manner. Our data indicate that these transcripts, including the liver sialyltransferase mRNA, are derived from a single genetic locus. Furthermore, we report an instance of apparent alternate splicing within the sialyltransferase coding region that predicts a divergent kidney-specific polypeptide. Implications of these findings will be discussed.

MATERIALS AND METHODS

RNA Preparation and Analysis-Total RNA from rat tissues (Spra- gue-Dawley) was extracted by the guanidinium isothiocyanate method (15, 16). Blot analysis (18) was performed after fractionation of the RNA in formaldehyde-agarose gels (17). Transcript protection analysis was conducted as described previously (19, 20). Hybridizations were conducted at 57 “C, subsequently digested with 200 units of S1 nuclease for 60 min (25 “C), and resolved on denaturing 5.5% poly- acrylamide gels.

Probe Preparation-The probes for hybridization of sialyltransferase mRNA were generated from a partial rat a-2,6-sialyltransferase cDNA sequence (13). The 780-base pair BstEII fragment (see Fig. 1, positions +495-1269) represents the distal two-thirds of the coding sequence. The 3”untranslated region probe was generated from a 930-base pair restriction fragment bordered by a BstEII site (position +1745) at its 5’-end (see Fig. 1). Gel-purified fragments were uniformly labeled with [a-32P]dATP by the random primer method (21). Probes for differential RNA blot analysis (Fig. 6, A and B) were generated using antisense oligonucleotides designed to recognize selectively transcripts representing alternate forms of rat kidney cDNA clones (14). P1 (5’-CAGACACACTGAGAATACGAGTGAGGC-3’) and P5 (5’-TCTCTCGACCAAGC-3’), antisense primers that hybridize to regions immediately upstream of the divergent border and specific for RKA and RKE/RLA, respectively, were used to generate uniformly labeled extension probes from respective RKA and RKE single-stranded templates in M13. The resulting probes contain 180 and 234 nucleotides complementary to regions immediately 5’ of the divergence site of RKA and RKE/RLA, respectively (see Fig. 5A). For transcript protection analysis, restriction fragments isolated from a partial rat a-2,6-sialyltransferase cDNA clone spanning either 125 or 560 nucleotides of coding information 5’ of the +495 BstEII or +934 BglII site, respectively, were 5’-end-labeled with polynucleotide kinase (19).

DNA Isolation and Analysis-Restriction digests of rat genomic DNA were subjected to blot analysis (19) and probed with either the radiolabeled coding region or the 3”untranslated region of the hepatic a-2,6-sialyltransferase (18). A rat kidney X g t l l cDNA library (Clon- tech Laboratories) was screened using the 780-base pair BstEII hepatic a-2,6-sialyltransferase coding probe. Twenty-four positively

17389

17390 Sialyltransferase Expression

t“l 100 nt

e R B E P I T I T T P/B BE

i T T BE B

’’ ATG I TGA 3’

+1 I 6 A 96 1212 I i HA

k“--- FIG. 1. Partial restriction map of rat hepatic a-2,6-sialyltransferase cDNA. The schematic represen-

tation, drawn to scale, illustrates a 1209-nucleotide (n t . ) open reading frame (hatched box) initiating at position +1 (ATG) followed by an extensive 3”untranslated tail. The cross-hatched box spanning nucleotides +27-78 and the upper arrowhead at position +189 denote the putative membrane-spanning domain and the amino-terminal end of the soluble form of the a2,6-sialyltransferase, respectively (1). The lower arrowhead at position +696 defines the junction between the divergent (RKA) and consensus (RKE) sequences in kidney (see Fig. 5). P, PstI; R, Eco RI; B , BglII; BE, BstEII. 780 and 930 denote the map position of the coding domain and 3”untranslated region probes, respectively, described under “Materials and Methods.”

hybridizing clones were plaque-purified (19) from IO6 recombinants. Select fragments were subcloned into M13 and sequenced using the dideoxy chain termination protocol (22).

RESULTS AND DISCUSSION

Tissue-Specific Expression of Sialyltransferase mRNA-A schematic representation and partial restriction map of the rat hepatic cu-2,6-sialyltransferase cDNA is depicted in Fig. 1. The full-length message is approximately 4.0 kb’ and contains P 1209-nucleotide open reading frame that is followed by a long 3”untranslated sequence (1,13). To assess tissue-specific differences in the expression of this message, RNA from various rat tissues was analyzed on Northern blots. Fig. 2A displays the results when a 780-base pair BstEII fragment representing the distal two-thirds of the hepatic sialyltrans- f e rae coding region was used as probe. As shown, submaxillary gland, kidney, lung, spleen, and liver (Fig. 2.4, lunes 1-5, respectively) generally maintain higher steady-state sialyltransferase mRNA levels; much lower levels are found in heart and brain (Fig. 2 A , lunes 6 and 7, respectively). In addition to differences in the level of sialyltransferase mRNA, there are also unexpected qualitative differences. At least three distinct mRNAs, ranging from 3.4 to 4.3 kb, are detected with the coding region probe. Whereas the predominant sialyltransferase mRNA in liver is the 4.0-kb form (Fig. 2 A , lune 5 ) , a larger, -4.3-kb form is expressed in other tissues. Among these tissues, sialyltransferase mRNA composition is most striking in kidney because, in addition to the larger 4.3-kb form, a much smaller form of -3.4 kb is also present (Fig. 2A, lane 2 ) .

The multiple RNAs may represent related species with sufficient sequence similarity to hepatic a-2,6-sialyltransferase to cross-hybridize to the coding region probe (transcripts coding for other sialyltransferases, for example). However, because of the stringent hybridization and washing conditions (68 “C) that were employed, probe cross-hybridization should be limited to transcripts that share high degrees of homology. To assess the relationship of the multiple transcripts, a 930- nucleotide cDNA probe representing a portion of the 3’-

’ The abbreviation used is: kb, kilobase(s).

untranslated region of the hepatic sialyltransferase gene sequence was constructed. As shown in Fig. 2B, the 3”untranslated region probe recognized the multiple sialyltransferase forms even under high hybridization stringency. More impor- tant, identical RNA patterns were generated when coding or 3”untranslated regions were used as probes (Fig. 2, compare A and B).

Among the mechanisms that could account for the above observations, two are most plausible. The first possibility is that the multiple RNAs are transcribed from multiple genes and that these genes share close sequence conservation not only in the coding region, but also in portions of the untranslated region. As mentioned earlier, RNA blotting was performed under conditions that limit cross-hybridizations. If multiple genes are involved, then the sequence conservation, in the coding domain as well as in portions of the untranslated region, must be extremely extensive. Although conserved coding sequences among members of related genes are expected, the preservation of noncoding sequences is unusual. The second and more likely possibility is that some or all of the multiple transcripts are derived from a single gene sequence. Size heterogeneity can be the result of alternate transcriptional start sites, alternate termination, and/or alternate splicing (23).

Analysis of Genomic Sequences-To determine the com- plexity of sialyltransferase sequences in the genome, restriction digests of rat genomic DNA were hybridized with either the sialyltransferase coding (Fig. 3A) or 3“untranslated (Fig. 3B) probe. Because many of the restriction enzymes have recognition sites within the coding region as well as in the intervening sequences, a fairly complex pattern was generated when the blot was hybridized to the coding probe (Fig. 3A). In contrast, the 3’-untranslated probe hybridized to a single band in four of the restriction enzyme digests (Fig. 3B, lanes I , 2, 5, and 6). An additional minor band is observed in the BglII digest (Fig. 3B, lane 3 ) due to the presence of a BglII site within the 3’-untranslated region (see Fig. 1). This ob- servation indicates that the 3’-untranslated sequence resides in a unique genomic region (multiple restriction fragments

Sialyltransferase Expression 17391

5UG 10 UG FIG. 2. RNA blot analysis of rat 1 2 3 4 5 1 2 3 4 5 6 . 7

1

tissues with hepatic a-2.6-sialyl- A. transferase gene sequences. Either 5 or 10 pg of total RNA isolated from rat submaxillary gland, kidney, lung, spleen, liver, heart, and brain (lanes 1-7, respectively) was fractionated in 1.0% agarose, 2.2 M formaldehyde gels and electrotransferred to nylon membranes. Blots were hybridized with either hepatic a- B. 2,6-sialyltransferase coding probe ( A ) or 3”untranslated probe ( B ) as described under “Materials and Methods.”

n C r

k b

5.5 -

2.5 -

1.1 -

1 2 3 4 5 6 1 2 3 4 5 6

FIG. 3. Southern blot restriction analysis of rat genomic sialyltransferase sequences. Rat genomic DNA was digested to completion with EcoRI, NcoI, BglII, HaeII, BstEII, or PstI (lanes I - 6, respectively); resolved in 0.8% agarose gels; transferred to a nitro- cellulose membrane; and hybridized with either hepatic a-2,6-sialyltransferase coding probe ( A ) or 3”untranslated probe ( B ) as previously described.

are expected if the 3”untranslated region is represented in more than one locus) (24,25).

Transcript Protection Analysis of Sialyltransferase RNAs- To obtain additional evidence that multiple transcripts are generated from differential utilization of a unique a-2,6-sialyltransferase genetic region, we sought to define further the structural relationship between some of these transcripts. For this purpose, liver and kidney RNAs were chosen. As discussed earlier, kidney expresses a 4.3-kb mRNA and a 3.4-kb mRNA that are distinct in size from the hepatic sialyltransferase transcript. Fig. 4 depicts S1 nuclease protection analysis using ”P-end-labeled cDNA probes generated from a partial hepatic a-2,6-sialyltransferase cDNA clone. First, a probe engineered to protect a 125-nucleotide coding sequence lying 5‘ of a BstEII site (position +495 in Fig. 1) was used. As shown in Fig. 4A, liver and kidney mRNAs protected the predicted 125-nucleotide fragment. To extend the analysis of the a-2,6-sialyltransferase coding domain, a second S1 nuclease probe was generated to protect 560 nucleotides 5‘ of the BglII site (position +934 in Fig. 1). Although both liver and kidney RNAs protected the full-length 560-nucleotide

fragment, an additional protected fragment of 240 nucleotides was generated by kidney RNA (Fig. 4B). The data suggest a sequence divergence in a subpopulation of kidney sialyltransferase mRNAs and are consistent with the expression of at least two distinctly sized kidney transcripts. The point of divergence, based on S1 nuclease protection analysis, should be at approximately position +696, which lies between the BstEII (position +495) and BglII (position +934) sites. More- over, genomic sequence analysis indicates that position +696 is an intronlexon boundary with defined 5‘-splice donor and 3’-splice acceptor sequence information.* Altogether, the ex- isting data are consistent with an alternate splicing mecha- nism whereby divergent transcripts from a single sialyltransferase gene are generated. Consequently, kidney RNA should contain a population of sialyltransferase mRNAs that diverge from the hepatic sequence in the area 5’ of position +696.

Isolation of Kidney cDNA Clones Representing Divergent Forms of Hepatic Sialyltransferase-To substantiate the no- tion that some divergent forms of sialyltransferase mRNAs in kidney are generated by alternate splicing, we proceeded to screen a rat kidney cDNA library for sialyltransferase clones (see “Materials and Methods”). One positively hybridizing clone, RKE, contained an insert of approximately 2.35 kb and shared 100% nucleotide sequence homology with the distal 848 nucleotides of the cloned rat liver a-2,6-sialyltransferase (RLA) coding domain as well as 1.5 kb of identical 3”untranslated sequence information. Other positive isolates, exempli- fied by RKA, which contained a 2.24-kb insert, shared identical nucleotide sequence homology over the distal 515 nucleotides of coding and 1479 nucleotides of 3”untranslated information with both the RKE and RLA sialyltransferase cDNA sequences. However, RKA also contains a block of divergent sequence information of a t least 250 nucleotides in length. This divergent domain is 5’ to the common coding block with the junction mapping precisely to position +696. A partial comparative nucleotide sequence of rat liver a-2,6- sialyltransferase (RLA) and rat kidney cDNA clones (RKA and RKE) is shown in Fig. 5A. The arrowhead denotes position +696. The corresponding predicted peptide sequences are shown in Fig. 5B. It is quite obvious from this comparison that RKA transcripts predict a polypeptide that is divergent from hepatic a-2,6-sialyltransferase. Furthermore, approximately 1500 nucleotides of 3”untranslated sequence information are shared by all RL and RK clones examined (data not shown), and this is consistent with the ability of the 3‘- untranslated probe to hybridize to all forms of sialyltransferase RNAs on Northern blots (Fig. 2B).

Differential Northern Blot Analysis of Rat LiverlKidney

* X. C. Wang and J. T. Y. Lau, unpublished observations.


FIG. 4. Transcript protection analysis of rat tissue RNA with hepatic a-2,6-sialyltransferase sequences. Total RNA (15 pg) was isolated from rat liver (lanes 2 and 4 ) and kidney (lanes 3 and 5) and analyzed by S1 nuclease digestion with either a 5' end-labeled 125-nucleotide BstEII probe ( A ) or a 560-nucleotide BgfII probe ( B ) as described under "Materials and Meth- ods". Lanes I and 6 are control tRNA samples (50 pg) hybridized with the respective probes. Protected fragments were resolved in 8 M urea, 5.5% poly- acrylamide denaturaing gels alongside 5' end-labeled pBR322/HpaII molecular size standards. The arrowheads denote the size of protected transcript segments a t 125 nucleotides ( A ) and 560 and 240 nucleotides ( B ) .

RKE

RLA

RKA

RKE

RLA

RKA

RKE

RLA

RKA

RKE

RLA

RKA

RKE

RLA

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RLA

RKA

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309,

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G T C G T C T C T T C T G C A G G A T C C T G ~ C T C C C A G C T T T C A T G A T G

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TAATGAACTCTCAGTTAGTCACCACAGAAAAGCG~CCTCAAGGACAGTTTGTACACCG~GG

TAATGAACPCTCAGTTAGTCACCACAGAAAAGCGCTTCCTCAAGGACAGTTTGTACACCGAAGG

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CTAATTGTATGGGACCCATCCGTGTATCATGCAGATATCCCAAAGTGGTATCAG~CCAGACT

CTAATTGTATGGGACCCATCCGTGTATCATGCAGATATCCCAAAGTGGTATCAGAAACCAGACT

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A.

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B.

B. 4 5 6 (nt.)

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FIG. 5. Comparative nucleotide and peptide sequence analysis of rat kidney and liver sialyltransferase isoforms. Displayed is partial sequence information for two distinct classes of rat kidney (RKA/RKE) and liver (RLA) sialyltransferase cDNA clones. The arrowheads denote the mapped point of divergence between RKA and RKE/ RLA nucleotide (A, position +696) and peptide ( B , position +232) sequences.

Transcripts-To assess the relationship between the diver- domains from the RKA and RKE cDNA clones. Total cellular gent kidney RKA and RKE cDNA clones and the alternate RNA from rat liver (Fig. 6A, lane I ) or kidney (lane 2) was transcripts visualized on Northern blots, we engineered assessed by Northern blot analysis with probes specific for probes to recognize selectively either the common or divergent the RKA divergent domain (Fig. 6 A ) , the RKE/RLA-specific

Sialyltransferase Expression 17393

1 2 1 2 1 2

A B C FIG. 6. Differential Northern blot analysis of rat liver and

kidney sialyltransferase mRNAs. Total RNA (10 pg) extracted from rat liver (lane 1 ) or kidney ( l a n e 2) was fractionated in 1.0% formaldehyde gels and electrotransferred onto nylon membranes. RNA blots were differentially hybridized with uniformly labeled probes that selectively recognize the RKA divergent domain (A), the RKE/RLA-specific domain ( B ) , or a 780-base pair BstEII coding domain probe recognizing sequences common to all isoforms (C). See “Materials and Methods” for probe construction.

domain (Fig. 6B), or the common coding region that lies 3’ of the divergence point (Fig. 6C). When kidney RNA is probed for the RKA domain sequences (Fig. 6A, lane 2) , only the smaller 3.4-kb mRNA, but not the larger 4.3-kb mRNA, is visualized, suggesting that the RKA-type clones are derived from the 3.4-kb transcript. Furthermore, the relative abun- dance of the 3.4-kb mRNA (Fig. 6A, lane 2) indicates that the RKA transcripts constitute a major and significant proportion of sialyltransferase mRNA in kidney. A quite unexpected finding is the presence of RKA hybridizing sequences in liver (Fig. 6A, lane 1 ). However, the relatively low signal intensity (Fig. 6, compare A and B ) strongly suggests that a subpopulation of hepatic sialyltransferase mRNAs contain the RKA domain and that multiple RNAs contribute to the single band observed in liver RNA blots. Paradoxially, the hepatic transcripts that contain RKA-specific sequences are not detected in nuclease protection analysis using the 560-nucleotide BgllI probe (see Fig. 4B); the use of the S1 nuclease probe success- fully predicted the existence of RKA transcripts in kidney. The precise configuration and significance of these apparently divergent liver transcripts are unknown and currently under investigation.

A probe designed to recognize the RKE/RLA coding sequences immediately 5’ of the assigned divergence junction (position +696) was also used to assess sialyltransferase expression in liver and kidney transcripts (Fig. 6B). In addition to the expected signal in liver RNA (Fig. 6B, lane 1 ), it is quite clear that this domain is present in both size classes of kidney transcripts ( l a n e 2). One obvious interpretation is that RKE sequences situated upstream of the divergent border are represented in the RKA-type transcripts (ie. the smaller kidney transcripts). Given that the isolated RKA cDNA clones are less than full-length, this is a plausible explanation and consistent with a cassette-type model of alternate splicing (23, 38). Furthermore, additional alternate processing events must exist to account for the relatively small size of the 3.4- kb transcripts. The data suggest that the larger 4.3-kb kidney transcripts represent RKE class cDNA clones. This interpretation is based on (i) the presence of RKE upstream sequences in these transcripts (Fig. 6B, lane 2) and (ii) the inability of RKA-specific probes to hybridize to these messages (Fig. 6A,

lane 2). As mentioned earlier, our partial RKE-type cDNAs are colinear with the reported hepatic a-2,6-sialyltransferase sequence (refer to Fig. 5A). Although these findings suggest that the larger kidney transcripts may represent a hepatic sialyltransferase homolog, there persists an obvious discrep- ancy between the overall size of the respective transcripts that may be the result of additional processing events.

Together, our data document that multiple transcripts are generated in a tissue-specific manner from a common sialyltransferase gene sequence. We have demonstrated that one processing event operative in sialyltransferase expression is the tissue-specific alternate splicing at divergence point +696. It is clear, however, that this alternate splicing event cannot fully account for the heterogeneity displayed in sialyltransferase mRNAs and that additional mechanisms are operative. Alternate mechanisms, such as multiple transcriptional initiation sites (7, 32, 33), multiple transcriptional termination sites (34), and additional alternate splice sites (35, 36), are currently under investigation. The remote possibility that genomic rearrangement generates the multiple liver and kidney forms can be discounted; comparative genomic restriction analysis between liver and kidney found no evidence of rearrangement?

Generation of a family of transcripts from single genes has been reported in the expression of a large number of genes including calcitonin (26), myosin light chains (25,27,28), and 7-protein (29). Among the glycosyltransferases, multiple transcripts have been reported for mouse and bovine /3-1,4-galac- tosyltransferases (6, 7, 30) as well as for yeast GlcNAc-l- phosphoryltransferase (31). The functional significance of multiple transcripts remains unclear. In galactosyltransferase expression, alternate transcriptional initiation results in mRNAs that putatively use one of two in-frame AUGs for translational initiation (7). Based on our current understanding of membrane-spanning domains and protein topology (37), it was suggested tha the two forms of galactosyltransferase differ in their topological orientation within the Golgi membrane (7). Multiple polypeptides encoded by single genes are widely distributed (23, 38). An example is the troponin T gene in which alternate utilization of coding exons generate at least 10 unique mRNAs (39). Consistent with this heterogeneity is that antigenically distinct troponin T isoforms appear in different muscle types and at different stages of development (40-42). For proteins with multifunctional domains, structural polymorphism can be derived by multiple RNA processing pathways. Our data are highly suggestive of sialyltransferase polypeptide diversity as a result of alternate splicing.

Acknowledgments-We thank Dr. Peter Lance for helpful discus- sions and Marcia Held for preparation of the manuscript.

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