OST48 homologue, from Drosophila melanogaster

Gene, 158 (1995) 209-212 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

209

GENE 08890

PCR-mediated cloning and sequencing of the DmOST50 gene, a WBP1/AvOST50/OST48 homologue, from Drosophila melanogaster (Oligosaccharyltransferase; glycosylation; yeast; avian; canine; ribophorin; nucleotide sequence; comparison)

Igor Stagljar, Markus Aebi and Stephan te Heesen Mikrobiologisches Institut, ETH Zfirich, 8092 Zfirich, Switzerland

Received by G. Bernardi: 5 December 1994; Revised/Accepted: 23 January/30 January 1995; Received at publishers: 2 March 1995

SUMMARY

Oligodeoxyribonucleotides were used in a PCR reaction to amplify the conserved region of the DmOSTSO cDNA encoding an oligosaccharyltransferase subunit from Drosophila melanogaster (Din). The amplified fragment was cloned and sequenced, and was then used as a homologous probe to isolate a DmOSTSO cDNA from a kZAP library. The deduced amino acid (aa) sequence of DmOst50p shows 27.1% identity with the corresponding sequence of the yeast Wbplp, 62.4% identity with the avian AvOst50p and 62.7% with the canine Ost48p sequences. 17% of all aa residues were found to be identical among all species tested, indicating a high degree of conservation during evolution.

INTRODUCTION

Asparagine-linked glycosylation is an essential protein modification that takes pllace in the endoplasmic reticulum of all eukaryotic cells. The central step is the co-translational transfer of the core oligosaccharide which is preassembled on the lipid carrier dolichol phosphate, to selected Asn-Xaa-Ser/Thr residues of nascent polypeptide chains (Kornfeld and Kornfeld, 1985; Herscovics and Orlean, 1993). This reaction is catalyzed by the enzyme N-oligosaccharyltransferase (OST). In the yeast Saccharomyces cerevisiae, the two proteins Wbplp and Swplp have been identified as essential comCorrespondence to: Dr. S. te Heesen, Mikrobiologisches Institut, ETH Z~rich, SchmelzbergstraBe 7, CH-8092 Ztirich, Switzerland. Tel. (41-1) 632-3327; Fax (41-1) 632-1148; ,e-mail: [email protected]

Abbreviations: aa, amino acid(s); Av (Av),avian; bp, base pair(s); cDNA, DNA complementary to RNA; dNTP, deoxynucleoside triphosphate; Din, Drosophila melanogasiter; DmOST50, DNA encoding DmOst50p; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; OST, oligosaccharyltransferase; PCR, polymerase chain reaction; Pollk, Klenow (large) fragment of E. coli DNA polyraerase I; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaC1/0.015 M N%.citrate pH 7.6; TM, transmembrane, Xaa (X), any aa. SSDI 0378-1119(95)00172-7

ponents of the OST complex (te Heesen et al., 1992; 1993). Recently, the yeast OST complex has been purified, and shown to be a complex composed of four to six subunits, two of which were Wbplp and Swplp (Knauer and Lehle, 1994; Kelleher and Gilmore, 1994). Mammalian OST activity was purified as a complex consisting of ribophorins I and II, and a 48-kDa subunit (Ost48p) (Kelleher et al., 1992). The protein sequence of Ost48p was found to be 24.6% identical to yeast Wbplp. Regions of sequence identities between the two proteins were observed throughout the Ost48p sequence, rather than being restricted to a single conserved region (Silberstein et al., 1992). Purification of avian OST activity as a protein complex containing avian ribophorin I and II and a 50-kDa subunit has been reported (Kumar et al., 1994). The aa sequence of the 50-kDa component turned out to be 25% identical to yeast Wbplp and 92% identical to the sequence of canine Ost48p. In order to identify functionally important and therefore conserved residues of the Wbplp-subunit of the OST complex, we wanted to clone and to sequence homologous subunits from other species. In this report, we present the cloning and sequence of the WBP1/AvOST50/OST48 homologue from D. melanogaster (Din).

210 EXPERIMENTAL AND DISCUSSION

(a) Cloning and sequencing of D m O S T 5 0 eDNA The set of oligos used for PCR amplification was designed from the previously published 0 S T 4 8 eDNA sequence (Silberstein et al., 1992). The oligos were selected in regions of the aa sequence displaying a strong degree of identity between the canine Ost48p and the yeast Wbplp (te Heesen et al., 1991) (see legend to Fig.l). PCR amplification of Dm eDNA using primers H1 and H2 yielded a fragment of approx. 650bp (data not shown). After purification, the PCR product was treated with Pollk, phosphorylated with T4 polynucleotide kinase, cloned in the pBluescript KS vector, amplified in E. coli and sequenced (see region from nt 90 to 195 in Fig. 1). The corresponding deduced aa sequence was very similar to both the yeast Wbplp and canine Ost48p.

The amplified PCR fragment was used as a homologous probe to screen a XZAP library containing Dm eDNA. Three positive clones were purified by successive rounds of plaque isolation, and plasmid DNAs were isolated by in vivo excision. Analysis of the eDNA inserts in these plasmids revealed the presence of common sequences.

(b) Structural features of the Dm homologue of Wbp 1p/AvOst50p/Ost48p Sequence analysis of the isolated DmOST50 eDNA revealed a 1347-bp ORF encoding a putative protein of 449 aa (49.8 kDa) (Fig. 1). The ORF starts with two in-frame ATG codons. The consensus sequence surrounding the first ATG is compatible with a translation initiation signal (Kozak, 1989). Hydropathy analysis using the method of Kyte and Doolittle (1982) revealed the

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Fig. 1. Nucleotide sequence of DmOST50 eDNA and the deduced aa sequence. The predicted signal peptidase cleavage site between aa 18 and 19 is designated by an arrowhead (yon Heijne, 1986). The underlining of aa 412-431 designates a predicted TM segment. The stop codon is marked with an asterisk. Sequencing was performed with Sequenase Version 2.0 (US Biochemical) according to the recommended protocol for double-stranded templates (Del Sal et al., 1988), using either universal primers or specific synthetic primers. Sequence data have been deposited in the EMBL database under accession No. X81999. Methods: Dm cDNA was prepared as described in Sambrook et al. (1989). PCR was performed using primers H1 5'-GTGTGCGCCAGCGGCCCTCGCACCTTGGT (nt 85-114 of 0ST48 eDNA) and H2 5'-GGAGCCACTGAAGACGACCCGGGCATTGTT (nt 718-738 of 0ST48 eDNA) with an automated temperature cycling device (model 9600; Perkin-Elmer Cetus) with 0.5 M KC1/0.2 M Tris.HC1 pH 8.4/1 mM MgC12/I% Tween 20/0.05 mM each of the four dNTPs/0.5 ~tM of each primer/2.5 units of SuperTaq polymerase (Perkin-Elmer Cetus) and 0.5 ~tl of Dm eDNA in a total volume of 100 ~tl. After 11 min of incubation at 98°C, 35 of the following cycles were done: 95°C/1 min, 42°C/2 min, 72°C/3 rain. The PCR products were analyzed on a 0.8% agarose gel and extracted using QIAEX resin according to manufacturers protocol (DIAGEN, Germany). The DNA fragment was blunt-ended with Pollk and phosphorylated with T4 kinase before ligation into the unique SmaI site of the pBluescript KS(+) vector and was then used as a probe for screening a Dm XZAP eDNA library. Approx. 450 000 recombinant phages were screened using standard techniques with a eDNA probe prepared by random priming. Hybridization of the probe was done in 50% formamide/6% dextransulfate/1 M NaC1/0.1% SDS at 42°C for 16 h. Filters were washed once for 15 min at room temperature in 2 x SSC/0.1% SDS and twice for 30 min at 65°C in 2 x SSC/0.1% SDS.

211 a yeast invertase-Wbplp fusion, the invertase chimera was retrieved from the early Golgi compartment to the endoplasmic reticuhim in yeast, indicating a functional ER-retrieval sequence within the DmOst50p C terminus (Gaynor et al., 1994). The percentage of sequence identity for insect DmOst50p and yeast Wbplp is 27.1%. 62.7%/62.4% identical aa were detected by comparing DmOst50p with canine Ost48p/avian AvOst50p, using the Bestfit program. Ost48p and AvOst50p share 92% identical residues. In order to establish equal conditions for all comparisons, only the predicted mature sequences were compared. The leader sequences of Wbplp/ AvOst50p/Ost48p do not show similarity (not shown). A comparison of all sequences is shown in Fig. 3. There are conserved blocks throughout the sequence, separated by small gaps of insertions/deletions. 73 aa of a compared region of 446 (17%) were conserved among all species tested. The most conserved region is found between position 221 and 246 of the DmOst50p sequence, with 11 identical aa out of 26 (consensus GX4LX4QX2. NNARXVX2GSX 2 F). A search of Swissprot and PIR databases using this consensus sequence yielded only the OST subunits themselves, even if three mismatches were allowed. The functional significance of this sequence is demonstrated by the finding that in the yeast wbpl-I mutation the NNAR motif is mutated to NNAC (S.teH.

presence of two major hydrophobic segments (Fig. 2). The N-terminal hydrophobic segment located between aa 1 and 18 resembles ~L cleavable signal sequence for initiating TM translocation of the endoplasmic reticulum (von Heijne, 1983; 1986). The signal peptidase processing site in DmOst50p is predicted to be located between aa 18 and 19 (index 7.5), usi:ag the weight-matrix method of von Heijne (1986). A see,and nonpolar segment between residues 412 and 431 (Figs. 1 and 2) is compatible with a TM domain of the protein. The hypothetical protein does not contain N-linke, d glycosylation consensus sites (NXS/T). The C terminus of the protein is characterized by two Lys residues at positions - 3 and - 4 (Fig. 1), a signal which mediates retrieval of type-I TM proteins from the Golgi to the endoplasmic reticulum of higher eukaryotic cells (Jackson et al., 1990; Shin et al., 1991). When the 20-aa C-terminal cytoplasmic tail sequence of DmOst50p protein was used to replace the C terminus of I

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Fig. 2. Hydropathy plot of DmOst50p according to Kyte and Doolittle (1982), using a 9-aa averaging window.

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Fig. 3. Comparison of DmOst50p sequence with the yeast Wbplp, avian AvOst50p and canine Ost48p. The gaps introduced to maximize the homology are represented by dashes. Those aa common to at least two sequences are visualized in reverse contrast. Methods: Sequence alignments were performed using the Clustal method of the MegAlign program (DNA Star, Madison, WI, USA). The parameter used for multiple alignment was 10 for both Gap penalty and Gap length penalty. For determination of percentage identity, the Bestfit program (gap weight 3.000, length weight 0.100) of the Genetics Computer Group (Madison, WI, USA) Package 7.3 was used. Only the mature proteins without the predicted leader sequence were compared.

212 and M.A., unpublished results). Clearly, further mutational analysis will be required to evaluate the specific function of this sequence.

(c) Conclusions (1) A cDNA (DmOST50 cDNA), a WBPlp/AvOst50p/Ost48p homologue, has been isolated from Din. The DmOST50 cDNA contains an ORF of 1347 bp, encoding 449 aa (49.8 kDa). (2) DmOst50p is a type-I TM protein, containing a potential leader peptide sequence and a TM region close to the C terminus. (3) The DmOst50p shows 27% identity to yeast Wbplp, and 62% identity to avian AvOst50p and canine Ost48p. 17% of the residues are conserved among all species tested, indicating a high degree of evolutionary conservation of the oligosaccharyltransferase subunit.

ACKNOWLEDGEMENTS

We thank Dr. M. Noll and Dr. O. Zilian for Dm total RNA and kZAP library, Dr. M. Kertesz for careful reading of the manuscript and Dr. C. Weissmann for his support. This work was supported by grant 3100-040350.94/1 from the Swiss National Science Foundation.

REFERENCES Del Sal, G., Manfioletti, G. and Schneider, C.: A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res. 16 (1988) 987. Gaynor, E., te Heesen, S., Graham, T., Aebi, M. and Emr, S.D.: Signalmediated retrieval of a membrane protein from the golgi to the ER in yeast. J. Cell Biol. 127 (1994) 653-665.

Herscovics, A. and Orlean, P.: Glycoprotein biosynthesis in yeast. FASEB J. 7 (1993) 540-550. Jackson, M.R., Nilsson, T. and Peterson, P.A.: Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J. 9 (1990) 3153 3162. Kelleher, D.J. and Gilmore, R.: The Saccharomyces cerevisiae oligosaccharyltransferase is a protein complex composed of Wbplp, Swplp and four additional polypeptides. J. Biol. Chem. 269 (1994) 1-10. Kelleher, D.J., Kreibich, G. and Gilmore, R.: Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorin I and II and a 48 kd Protein. Cell 69 (1992) 55-65. Knauer, R. and Lehle, L.: The N-oligosaccharyltransferase complex from yeast. FEBS Lett. 344 (1994) 83-86. Kornfeld, R. and Kornfeld, S.: Assembly of the asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54 (1985) 631-664. Kozak, M.: The scanning model for translation: an update. J. Cell Biol. 108 (1989) 229-241. Kumar, V., Heinemann, F.S. and Ozols, J.: Purification and characterization of avian oligosaccharyltransferase. J. Biol. Chem. 269 (1994) 13451 13457. Kyte, J. and Doolittle, R.F.: A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157 (1992) 105-132. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Shin, J., Dunbrack, R.L., Lee, S. and Strominger, J.L.: Signals for retention of transmembrane proteins in the endoplasmic reticulum studied with CD4 truncation mutants. Proc. Natl. Acad. Sci. USA 88 (1991) 1918-1922. Silberstein, S., Kelleher, D.J. and Gilmore, R.: The 48-kDa subunit of the mammalian oligosaccharyltransferase complex is homologous to the essential yeast protein WBP1. J. Biol. Chem. 267 (1992) 23658 23663. te Heesen, S., Janetzky, B., Lehle, L. and Aebi, M.: The yeast WBPI is essential for oligosaccharyltransferase activity in vivo and in vitro. EMBO J. 11 (1992) 2071-2075. te Heesen, S., Knauer, R., Lehle, L. and Aebi, M.: Yeast Wbplp and Swplp form a protein complex essential for oligosaccharyl transferase activity. EMBO J. 12 (1993) 279-284. te Heesen, S., Rauhut, R., Aebersold, R., Abelson, J., Aebi, M. and Clark, M.W.: An essential 45 kDa yeast transmembrane protein reacts with anti-nuclear pore antibodies: purification of the protein, immunolocalization and cloning of the gene. Eur. J. Cell Biol. 56(1991) 8-18. von Heijne, G.: Patterns of amino acids near signal-sequence cleavage sites. Eur. J. Biochem. 133 (1983) 17-21. von Heijne, G.: A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14 (1986) 4683-4690.