©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Expression of cDNA Encoding the Rat UDP-N-Acetylglucosamine:-6-D-Mannoside -1,2-N-Acetylglucosaminyltransferase II (*)

Giacomo A. F. D'Agostaro (1)(§)(¶), Alessandra Zingoni (1), Robert L. Moritz , Richard J. Simpson , Harry Schachter (2), Brad Bendiak (¶) (3)(**)

From the (1)Laboratory of Biophysics, ENEA, Casaccia 00060, Roma, Italy, theJoint Protein Structure Laboratory, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute, Melbourne 3050, Australia, the (2)Department of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8, and (3)The Biomembrane Institute and University of Washington, Seattle, Washington 98119

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

UDP-N-acetyl-D-glucosamine:-6-D-mannoside -1,2-N-acetylglucosaminyltransferase II (EC 2.4.1.143) (GnT II) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the enzyme purified from rat liver revealed a polypeptide of 42 kDa. Amino acid sequences were obtained from the N terminus and a tryptic peptide. Overlapping cDNA clones coding for the full-length rat GnT II were obtained. The complete nucleotide sequence revealed a 1326-base pair open reading frame that codes for a polypeptide of 442 amino acids, including a presumptive N-terminal membrane-anchoring domain. The region of cDNA coding for the C-terminal 389 amino acids of rat GnT II was linked in frame to a cDNA segment encoding the cleavable signal sequence of the human interleukin-2 receptor and transiently expressed in COS-7 cells. A 77-fold enhancement of GnT II activity over a control carrying the GnT II cDNA out-of-frame was detected in the culture medium at 72 h after transfection. H-NMR spectroscopy confirmed that the oligosaccharide synthesized in vitro by the recombinant enzyme was the product of GnT II activity. These data verify the identity of the cloned GnT II cDNA and demonstrate that the C-terminal region of the protein includes the catalytic domain.


INTRODUCTION

The identification of the oligosaccharide ligands that interact with cell adhesion proteins of the selectin family (1, 2) has lent direct experimental support to the model that cell surface carbohydrates act as carriers of specific information in cell interaction and recognition. Further evidence supporting this model is provided by the observation that the binding properties of neural cell adhesion molecule(3) , intercellular adhesion molecule(4) , and myelin Po protein (5) appear to be modulated by stage- or tissue-specific patterns of glycosylation. The precise changes of cell surface carbohydrates that occur during development, differentiation, and maturation (6) suggest that the expression of the enzymes that act in the biosynthetic pathway of complex carbohydrates is finely regulated and point to the need for elucidation of the molecular mechanisms of this control. Moreover, in vivo disruption by gene targeting of the 1,2-N-acetylglucosaminyltransferase I gene(7, 8) causes arrest of the development of mouse embryos at an early post-implantation stage, indicating that the glycosylation enzymes play a vital role in mammalian development. Consistent with this notion, defects of the activity of glycosylation enzymes have been implicated in a few rare human diseases, including I-cell disease(9) , congenital dyserythropoietic anemia HEMPAS(10) , and carbohydrate-deficient glycoprotein syndrome type II(11) .

The branching pattern of N-linked complex carbohydrates is specified by the action of distinct N-acetylglucosaminyltransferases, each one of which catalyzes the transfer in a specific glycosidic linkage of a GlcNAc residue from the donor substrate UDP-GlcNAc to a mannose residue of the trimannosyl core pentasaccharide(12) . The enzyme UDP-N-acetyl-D-glucosamine:-6-D-mannoside -1,2-N-acetylglucosaminyltransferase II (GnT II)()catalyzes the transfer of a GlcNAc residue from the donor substrate to the 6-linked mannose of the core pentasaccharide, forming a 1,2 glycosidic linkage(12, 13, 14) . This reaction, which initiates the formation of biantennary N-linked oligosaccharides, is an essential step in the biosynthetic pathway, leading from high mannose to complex N-linked oligosaccharides. In vitro enzymic studies indicate that the activities of GnT V and GnT VI, which initiate formation of the branches linked -1,6 and -1,4, respectively, to the 6-mannose of the trimannosyl core, require the prior action of GnT II(12, 15) . The physiological acceptor substrate of GnT II is generated by the prior action of Golgi -mannosidase II, which removes two -linked mannoses to expose the 6-mannose of the core pentasaccharide(12) . The activity of GnT II is markedly reduced in the mononuclear cells from at least one HEMPAS patient (10) and in cultured fibroblasts from two individuals affected by the recently identified carbohydrate-deficient glycoprotein syndrome type II(11) , as indicated by the results of enzyme assays and structural analysis of N-linked oligosaccharides. Distinct genetic mutations appear to occur in HEMPAS patients, as defects in the activities of different enzymes involved in the biosynthetic pathway of N-linked complex carbohydrates have been observed in these patients(10) . A defect of -mannosidase II activity that results from a reduced level of the -mannosidase II mRNA has been described in one HEMPAS patient who shows a normal level of GnT II activity(16) . The identification of the nature and origin of the genetic mutations underlying defects of GnT II activity in human diseases will contribute to the elucidation of the molecular mechanisms that regulate expression of the glycosyltransferases and processing enzymes involved in the biosynthetic pathway and thereby determine the classes of complex carbohydrates displayed at the cell surface.

As an initial step to characterize the transcription unit encoded by the GnT II gene and study the molecular mechanisms that regulate expression of the GnT II enzyme, the cDNA coding for rat GnT II was cloned and characterized. In this paper, the complete nucleotide sequence of overlapping cDNA clones that encode the entire GnT II polypeptide is reported. Analysis of the deduced primary structure indicates a domain architecture and a type II transmembrane topology similar to that of other glycosyltransferases. Transient expression in COS cells of the cDNA segment coding for the C-terminal region of rat GnT II was shown to direct the synthesis of an enzymatically active polypeptide, providing unambiguous verification of the cloned rat GnT II cDNA.


EXPERIMENTAL PROCEDURES

Purification of Rat Liver GnT II Polypeptide

The GnT II enzyme was purified from rat liver homogenates with major modifications of a procedure previously published (13) to increase yields. The procedure was scaled up 5-fold, using 3.75 kg of rat livers. It was found that step 3, using hydroxylapatite(13) , was difficult to scale up due to channeling in the column and the need to concentrate large volumes. Step 3 was replaced with a large Affi-Gel Blue column (15 2 cm), which was loaded, washed, and eluted with buffers as previously described in step 8 (excluding glycerol), in the same relative proportions as compared with the column volume. Step 5 (affinity chromatography on 5-Hg-UDP-GlcNAc-thiopropyl-Sepharose with NaCl elution) was modified by loading and washing with buffer M, containing 25 mM NaCl instead of 50 mM. Step 6 (affinity chromatography on 5-Hg-UDP-GlcNAc-thiopropyl-Sepharose with UDP-GlcNAc elution) was eliminated, as was step 8. Steps 7 and 9 (EDTA elutions from 5-Hg-UDP-GlcNAc-thiopropyl-Sepharose) were applied sequentially, using 25 mM NaCl in buffer M, instead of 50 mM, and adding MnCl to 25 mM after the first elution with EDTA to ensure excess MnCl was present in the loading buffer of the second column. Four additional chromatographic separations were performed to isolate the pure protein.

The sample from the third affinity chromatography step was loaded on a CM-Sephadex C-50 column (1.4 4.0 cm), equilibrated in 25 mM NaCl, 10 mM EDTA, 20% glycerol, 0.02% sodium azide, 0.1% Triton X-100, 25 mM MES, pH 6.8 (buffer X). Fractions (5.0 ml) were collected, using siliconized glassware(13) . The column was washed with 15 ml of the same buffer after loading, then with 100 ml of a solution containing 10 mM EDTA, 0.1% Triton X-100, 0.02% sodium azide, 20% glycerol, and 25 mM TAPS, pH 8.9. The column was then washed with 50 ml of a solution having the same constituents except that 50 mM CAPS, pH 10.2, instead of 25 mM TAPS, pH 8.9, was used. The enzyme eluted, essentially quantitatively, at pH 8.9 in six 5.0-ml fractions. Additional protein eluted at pH 10.2, but almost no GnT II activity was detected. This step gave approximately a 2-fold purification.

Active fractions from the pH-pulsed CM-Sephadex column were pooled and directly loaded on a small hydroxylapatite column (Bio-Gel HTP, Bio-Rad, 1.4 3 cm, washed with buffer X, described above). The column was washed with 15 ml of the same buffer, then with 50 ml of buffer G(13) , and 50 ml of buffer H(13) , both buffers having an additional 20% (v/total v) glycerol, collecting 5.0-ml fractions. The enzyme eluted with buffer G (0.1 M in phosphate) and could be monitored using 1-µl aliquots diluted to 20 µl with a solution containing 125 mM MES, 25 mM MnCl, and 1 mg/ml bovine serum albumin, pH 6.5. This step gave a 1.5-fold purification.

Active fractions (12, 13, 14, 15) from the hydroxylapatite column were pooled, dialyzed against buffer X, and loaded on a column of reactive yellow 86-agarose (Sigma, product R 8504, 1.5 3.0 cm, washed with 2.0 M NaCl, then with buffer X). 5-ml fractions were collected. The column was washed with an additional 40 ml of buffer X, then with 25 ml of the same buffer containing 0.5 M NaCl. The enzyme did not bind strongly to the reactive yellow-agarose but was retarded, eluting in the wash with buffer X. Some additional protein came off in the 0.5 M NaCl wash. This step gave a 1.3-fold purification of the active enzyme.

At this stage, the enzyme was essentially pure. It was subjected to gel filtration on a second, smaller Sephadex G-200 (Pharmacia Biotech Inc., Superfine) column (as in step 4, Ref. 13, except the column dimensions were 1.5 48 cm). A single peak of GnT II activity was observed. SDS-PAGE analysis of all active fractions showed a 42-kDa, silver-stained band, which correlated in intensity with enzymatic activity. Heavy loading of the gel showed a minor band of approximately 34 kDa.

Micropreparative Isolation of Protein and Peptides for Sequencing

Automated Edman degradation was performed using Applied Biosystems sequencers (470A and 477A). For sequence analysis of the intact protein, the GnT II enzyme was first desalted and purified free of detergent contaminants by reversed-phase HPLC through trace enrichment onto Brownlee RP-300 (30 4.6 mm and 30 2.1 mm) columns (17, 18). The concentrated sample was applied directly onto a Polybrene-treated glass fiber filter. For sequencing the 42-kDa polypeptide, the HPLC-purified enzyme was reduced (dithiothreitol) and alkylated (iodoacetic acid). The 42-kDa band was isolated by preparative SDS-PAGE (12% gel) and electroblotted onto a polyvinylidene difluoride membrane, which was sandwiched between the glass block and the Polybrene-treated glass fiber filter. For analysis of internal sequences, the 42-kDa band was electroeluted using a modified electroelution apparatus(18, 19) . The eluted protein was digested with trypsin at an enzyme:substrate ratio of 1:20. Resultant peptides were isolated by microbore reversed phase HPLC on a Brownlee RP-300 (30 2.1 mm) column.

Construction and Screening of a Rat Liver cDNA Library

A cDNA library was constructed in gt11 (20) from rat liver poly(A)-enriched RNA, by priming reverse transcription with oligo(dT), as previously described(21) . 5 µg of poly(A) RNA was used for cDNA synthesis. The cDNA library contained 3.2 10 gt11 phages. Recombinant phages were propagated and screened following procedures, which have been described(21) . The hybridization was carried out at 38 or 65 °C, depending on whether degenerate synthetic oligonucleotide or DNA fragments were used as probes. DNA oligonucleotides were synthesized with an automated synthesizer (Applied Biosystems, 381A) using the phosphoramidite chemistry. The synthetic oligonucleotides were purified and end labeled with [-P]ATP (Amersham Corp., >6000 Ci/mmol) and polynucleotide kinase, as previously described(21) . DNA fragments were labeled with [-P]dCTP (Amersham, > 3000 Ci/mmol) and Klenow's fragment to a specific activity of >10 cpm/µg following the random priming procedure(20) .

5`-Extension of Rat GnT II cDNA

Complementary DNA was synthesized from rat liver poly(A)-enriched RNA by priming reverse transcription with the rat GnT II-specific oligonucleotide 5`-CCGTCTTTATCGACATTCCTCAGC-3`, designated R9P1R, that is complementary to a sequence in clone R9 (Fig. 2). 200 ng of poly(A) RNA and a RNase H reverse transcriptase (Stratagene, StrataScript) were used for first-strand cDNA synthesis. Following purification on a GlassMax spin column (Life Technologies, Inc.), a homopolymer of deoxycytidilic acid was added to the 3`-end of single-stranded cDNA, using terminal deoxynucleotidyl transferase. The reaction (20 µl) was carried out in10 mM Tris-HCl, pH 8.4, containing 25 mM KCl, 1.25 mM MgCl, and 200 mM dCTP. The C-tailed cDNA was amplified by PCR using a nested rat GnT II-specific oligonucleotide 5`-CAAAGTTCAACTGGTACACTAGGG-3`, designated R9P3R (Fig. 2), as a 3`-primer and a 36-mer DNA oligomer 5`-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3`, where I indicates inosine, as a 5`-primer. This DNA oligomer, designated ``anchor oligonucleotide,'' contains an adaptor region, which carries multiple restriction sites (underlined) and a sequence complementary to the homopolymeric tail. Inosine residues were introduced in the homopolymeric tract of G to lower the T of the DNA oligomer. The reaction (100 µl) was carried out in 10 mM Tris-HCl, pH 8.3, containing 50 mM KCl, 1.5 mM MgCl, 0.01% gelatin, 0.5 mM of each of the four dNTP, 20 pmol of each oligonucleotide primer, 2.5 units of Taq DNA polymerase (AmpliTaq, Perkin-Elmer Corp.), and 5 pg of C-tailed single-stranded cDNA for 30 cycles in a thermo cycler (Perkin-Elmer, model 480). Cycle conditions were as follows: 94 °C, 1 min; 56 °C, 2 min; and 72 °C, 2 min. PCR amplification was also carried out on glass capillary plugs (2.5 µl) of agarose gel (3% NuSieve and 1% Seakem, FTC) containing size-selected cDNA molecules. In these reactions, a 20-mer oligonucleotide 5`-GGCCACGCGTCGACTAGTAC-3` representing the adaptor region of the anchor oligonucleotide was used as 5`-primer and either the GnT II-specific oligonucleotide R9P3R or R9P4R (Fig. 2) as a 3`-primer. PCR amplification was for 25 cycles, with the following conditions: 94 °C, 1 min; 60 °C, 2 min; and 72 °C, 2 min.


Figure 2: 5`-Extension of rat GnT II cDNA. The nucleotide sequence of the 5`-end of the open reading frame in gt11 cDNA clones R7, R9, and R9.5 (Fig. 1), including the artificial EcoRI site (lowercaseletters), is reported in the upperpart of the figure. The arrows indicate the 5`-3` direction and length of the three nested GnT II-specific oligonucleotides used as 3`-primers in first-strand cDNA synthesis and amplification. The analysis of the two major cDNAs generated by priming reverse transcription of rat liver poly(A) RNA with the GnT II-specific oligonucleotide R9P1R is illustrated in the lowerpart of the figure. The two cDNA species of 360 and 250 bp were size-selected by gel electrophoresis and amplified by PCR. LanesA and B, 360 bp of cDNA; lanesC and D, 250 bp of cDNA. The adaptor oligonucleotide was used as a 5`-primer and either the GnT II-specific oligonucleotide R9P3R or R9P4R as a 3`-primer. The PCR products were resolved by electophoresis through a 4% agarose gel and visualized by ethidium bromide staining and UV irradiation. A ladder of DNA fragments, which differ in length by 126 bp (lanem), was used as molecular size markers.



cDNA Subcloning and Nucleotide Sequencing

Restriction fragments of cDNA inserts were isolated from purified phage DNA and subcloned into the vectors pBlueScript SK or pBSKS and their variants plus and minus (Stratagene) by standard methods(20) . The PCR-amplified cDNA was cloned into the pCRII vector (Invitrogen). Escherichia coli strains XL1Blue or DH5Mcr (20) were used as plasmid carriers. DNA sequence analyses were performed either on the gt11 phage vector or on the pBR322-based vectors by the dideoxy chain termination method(22) , using -[thio-S]dATP (DuPont NEN, 1000-1500 Ci/mmol). Sequenase 2 (U. S. Biochemical Corp.) or Vent Exo (New England Biolabs) DNA polymerases were used in the sequencing reactions, according to the manufacturer's instructions. Sequences were determined on both strands, following the strategy outlined in Fig. 1. Nucleotide sequence data were analyzed on a DEC VAX/9000 computer, using the software developed by the Genetic Computer Group (GCG, Madison, WI)(23) . The hydropathy plot was generated using a GCG implementation of the Kyte and Doolittle algorithm(24) . Sequence data base searches were performed on the GenBank data base (release 84) and Protein sequence data base (SwissProt, release 29), using a GCG implementation of the Lipman and Pearson algorithm (25) or the BLAST programs(26) .


Figure 1: Restriction map and sequencing strategy of rat GnT II cDNA. The sequence corresponding to the coding region is indicated by the openbox. Only restriction sites relevant to the sequence analysis are indicated. The letter ``P'' denotes the position of the synthetic oligomer used as a specific primer in cDNA synthesis. Horizontalarrows indicate direction and extent of sequence determination. The arrows originating in a solidcircle represent sequences determined using the gt11 forward or reverse sequencing primers. The arrows originating in a solidsquare represent sequences determined from subcloned fragments using the KS, SK, T3, T7, or M13 reverse sequencing primers. The arrows originating in a solidtriangle represent sequences determined using GnT II-specific oligonucleotides.



Construction of Variable Reading Frame Expression Plasmids

The vectors used for expressing the rat GnT II cDNA were derived from pBC12BI (27) and kindly provided by Jarema Kochan (Dept. of Genetics, Hoffman-LaRoche Inc., Nutley, NJ). The 700-bp BamHI-SmaI fragment of pBC12BI containing the coding sequences and the large intron of the rat preproinsulin II gene (28) was replaced, by standard procedures(20) , with a 850-bp HinfI-NciI fragment of human cDNA for the IL-2 receptor, coding for the cleavable signal peptide sequence and the N-terminal region of the mature polypeptide(29) . The resulting plasmid denominated pLJ79 was digested with BstEII, blunt ended by filling-in, and digested with SacI to remove a SacI-BstEII fragment containing the human cDNA coding for the mature polypeptide of the IL-2 receptor. The large fragment of pLJ79 was recircularized by ligation to each one of three partially double-stranded synthetic DNA oligomers, whose 5`-ends are complementary to a SacI 3`-overhang. The sequences of the three DNA oligomers designated RF1, RF2, and RF3 are 5`-AGCTCCAGCTGAATGAATGA-3`, 5`-AGCTCGCAGCTGAATGAATGA-3`, and 5`-AGCTCAGCTGAATGAATGA-3`. These DNA oligomers contain a PvuII restriction site (underlined), which allows the insertion of a DNA fragment in all three reading frames, and is flanked by multiple translation termination codons. The vectors containing the DNA oligomers RF1, RF2, and RF3 were designated pLJ268, pLJ269, and pLJ267, respectively.

Plasmid pR9 containing the EcoRI fragment of gt11 clone R9 (Fig. 1) was digested with EcoRI and treated with mung bean nuclease (New England Biolabs). The blunt-ended EcoRI fragment from plasmid pR9 was purified by electroblotting onto and elution from a DEAE-membrane (Schleicher & Schuell, NA45) and digested with XmnI to remove the 3`-untranslated region of rat GnT II cDNA (Fig. 1). The 1176-bp EcoRI-XmnI fragment containing the coding sequences of rat GnT II cDNA, including the translation termination codon, was purified as described above and cloned into the unique PvuII site of vectors pLJ268 and pLJ269. The recombinant plasmids were designated pLJ268_R9 and pLJ 269_R9, respectively, and their structure was verified by digestion with several restriction enzymes and sequence analysis using multiple GnT II-specific oligonucleotides as sequencing primers. The sequence analysis using as a primer the rat GnT II-specific oligonucleotide designated N5 that represent the complement of a segment (nucleotide position 364-348 in Fig. 3) of the rat GnT II cDNA confirmed that in plasmid pLJ 268_R9 the rat GnT II cDNA is linked in frame to the cDNA for the signal sequence of the human IL-2 receptor, whereas in plasmid pLJ269_R9 the rat GnTII cDNA is out of frame, and a premature stop codon is generated (Fig. 4).


Figure 3: cDNA and predicted protein sequence of rat GnT II. Nucleotides and amino acids are numbered at the left of each line. The predicted protein sequence is shown in the standard three letter code below the cDNA sequence. Regions of the GnT II polypeptide from which direct protein sequence data were obtained are underscored with a solidbox. Openareas denote unidentified amino acids by protein sequencing. Asterisks indicate potential sites for N-linked glycosylation. The proposed membrane-anchoring sequence is underlined. The entire sequence shown was derived independently from both strands of the cDNA, as indicated in Fig. 1. Nucleotides 1-348 derive from clones p5R7, p5R13, p5R16, p5R27, and p5R32; 268-2155 from clones R7 and R9; 342-2104 from clone R9.4; 678-2155 from clone R9.3; and 914-2155 from clone R9.2. Where sequences from two or more clones overlapped, they were identical. The sequence in clone R9 complementary to the synthetic DNA oligomer used as a specific primer in the cDNA synthesis is underlined.




Figure 4: Structure of expression plasmids. These plasmids contain the SV40 origin of replication and a segment of human cDNA coding for the cleavable signal peptide sequence of the IL-2 receptor (hum il2_R sps) under transcriptional control of the Rous sarcoma virus long terminal repeat (RSVLTR). The rat cDNA region coding for the C-terminal 389 amino acids of GnT II is linked in frame (pLJ268_R9) or out of frame (pLJ269_R9) to the hum il2_R sps cDNA. The rat GnT II cDNA is flanked on its 3`-end by the 3`-untranslated region of the rat preproinsulin II gene (rat ins-2 3`UTR). The region of the rat preproinsulin II gene containing the first intron, including the donor and acceptor splice sites (rat ins-2 ivsA), flanks the 5`-end of the hum il2_R sps cDNA. The bacterial origin of replication and the -lactamase gene of the pLJ vectors are derived from pXF3 (27), a pBR322 derivative that lacks sequences inhibitory to DNA replication in COS cells. The nucleotide sequence of plasmids pLJ268_R9 and pLJ269_R9 at the junction between the hum il2_R sps and the rat GnT II cDNA was verified as described under ``Experimental Procedures'' and is reported in the lowerpart of the figure. The deduced amino acid sequence is shown in the three letter code below the nucleotide sequence. The opentriangle indicates the cleavage site of the signal peptidase.



Transfection of COS Cells

COS-7 cells were kindly provided by Paolo Comoglio (University of Torino) and grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum and 10 µg/ml gentamycin sulfate (complete medium), in a humidified atmosphere containing 5% CO, at 37 °C. Confluent cell monolayers were trypsinized, and 5 10 cell aliquots were plated in 60-mm dishes 24 h before transfection. COS-7 cells (80% confluent) were transfected either with plasmid pLJ268_R9 or pLJ269_R9, by liposome-mediated uptake, using Lipofectin (Life Technologies, Inc.) as described by the manufacturer. Culture media and cell monolayers were collected at 24, 48, and 72 h post-transfection and stored at -80 °C. As a control for transfection efficiency, COS-7 cells were transfected with pSV-neo and cultured in complete medium containing 600 µg/ml of G418 (Life Technologies, Inc., Geneticin). Colonies of neo cells were enumerated at 3 weeks after transfection. The transfection efficiency was 10%.

In Vitro Assay for GnT II Activity

Assays were carried out as previously described (13) with minor modifications. Instead of a glycopeptide, the reducing oligosaccharide having the structure Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc, referred to as MGn(+F) was used as an acceptor substrate. It was prepared from bovine fibrin as previously described (30) with two modifications. First, instead of direct treatment of the fibrin with hydrazine, glycopeptides were first prepared with Pronase (1/50) and were isolated on a Sephadex G-50 column. Second, re-N-acetylation was carried out directly after hydrazine treatment(31) . The H-NMR spectrum of the substrate is shown in panela of Fig. 5. Cell pellets (1 10 cells) were lysed in 80 µl of a buffer containing 50 mM NaCl, 15 mM MnCl, 1 mM sodium azide, 1% (v/v) Triton X-100, and 50 mM MES, pH 6.8. Culture media were diluted 4-fold with the above buffer. 20-µl aliquots of a cell lysate or diluted culture medium were used in each assay. Reactions (25 µl) were carried out in 50 mM MES, pH 6.8, containing 1 mM UDP-[C]GlcNAc, 465 µM MGn(+F), 40 mM GlcNAc, 15 mM MnCl, 50 mM NaCl, 1 mM sodium azide, and 1% (v/v) Triton X-100. The product was quantitated using small Dowex AG1-X8 columns(13) . Each assay was carried out in duplicate.


Figure 5: Identification of the product of recombinant GnT II catalysis by H-NMR spectroscopy. Structural reporter regions of 500 MHz H-NMR spectra of (a) the oligosaccharide acceptor and (b) the product catalyzed by recombinant GnT II secreted by COS-7 cells upon transfection with plasmid pLJ268_R9 are shown. Structures are shown above each spectrum, respectively. Indicated on the spectra are the anomeric proton signals ( = 4.4-5.3), the mannose H-2 signals ( = 3.95-4.3), the fucose H-5 and H-6 (methyl) signals, and the acetamido methyl signals ( = 2.0-2.1). Residues are numbered according to Vliegenthart et al. (43). Acetamido methyl protons are shown at 20-30% full intensity.



Identification of the Product of in Vitro Catalysis by H-NMR Spectroscopy

Samples containing the culture medium of COS-7 cells transfected with plasmid pLJ268_R9 at 72 h post transfection were concentrated approximately 4-fold and simultaneously dialyzed against a buffer containing 50 mM NaCl, 1.0% Triton X-100, 0.02% sodium azide, 15 mM MnCl, and 50 mM MES, pH 6.8, on a Micro-ProDiCon dialyzer (Bio-Molecular Dynamics), at 4 °C. This solution (2.0 ml) was incubated with MGn(+F) (300 nmol) and UDP-[C]GlcNAc (1 mCi/mmol, 10 µmol) at 37 °C for 75 min. After dilution with 10 ml of water, the reaction mixture was passed through two columns in tandem, followed by 5 40-ml washes with water, Dowex AG1-X8 (Cl form, 100-200 mesh, 2.5 8.2 cm), and Bio-Beads SM-2 (Bio-Rad, 20-50 mesh, 2.5 5.1 cm). The eluate was rotary evaporated and applied in a 3.0-ml volume to a Bio-Gel P-4 column (Bio-Rad, 200-400 mesh, 1.5 92 cm). Fractions (4 ml) were collected, and aliquots (10 µl) were monitored for C-labeled product. Two peaks were found, one centered at fraction 32, which comigrated with GlcNAc (25 nmol, based on specific activity) and one centered at fraction 21 (235 nmol). The high molecular weight material was concentrated and chromatographed on a Waters GlycoPak N HPLC column (7.8 300 mm), with acetonitrile/water (75/25) at 1.0 ml/min, monitoring at 200 nm. A major biphasic peak appeared at 125-145 min, which gave 225 mol of product. The product was examined in DO by 500 MHz H-NMR spectroscopy at 300 K on a Bruker AM-500 spectrometer (University of Washington). Chemical shifts were relative to internal acetone at = 2.225 ppm.

Northern Blot Analysis

Total RNA was prepared from rat organs as previously described(21) . Poly(A) RNA was selected by affinity chromatography on OligoTex-dT (Quiagen) according to the manufacturer's instructions. The RNA concentration of each preparation was determined from the absorbance at 260 nm(20) . 5-µg aliquots of each poly(A)-enriched RNA preparation were subjected to electrophoresis through an agarose gel containing 2.2 M formaldehyde(20) . RNAs of a defined length (Life Technologies, Inc., types II and III) were used as molecular size markers. Capillary transfer onto neutral nylon membranes (Stratagene, Duralon) was in 3.0 M NaCl, 0.3 M trisodium citrate (pH 7.0), for 16-18 h. The RNA blots were UV-irradiated at 1800 µJ to cross-link RNA to the membranes, which were prehybridized in a solution containing 50% (v/v) formamide (Life Technologies, Inc., UltraPure), 10% dextran sulfate, 1% SDS, 0.9 M NaCl, and 200 µg/ml of sheared salmon sperm DNA as a carrier at 42 °C for 4 h. The hybridization was carried out at 42 °C for 16-18 h in prehybridization solution containing a labeled DNA fragment at 2 10 cpm/ml. Membranes were washed three times at room temperature in 0.3 M NaCl, 30 mM trisodium citrate (pH 7.0), and 0.1% SDS and three additional times at 65 °C in 1.5 mM NaCl, 0.15 mM trisodium citrate (pH 7.0), and 0.1% SDS. Membranes were exposed to x-ray film (Kodak, XAR-5) at -80 °C, using two intensifying screens, for 24-72 h. Probes were stripped off a blot by washing the membranes once at 65 °C in water for 15 min and several times at 95 °C in 1.5 mM NaCl, 0.15 mM trisodium citrate (pH 7.0), and 0.1% SDS for 30-45 min.

The 5`-end probe was derived from plasmid p5R7 (Fig. 1) by digestion with EcoRI. The coding region probe was generated by PCR amplification of plasmid pR9 (see above) using as primers two GnT II-specific oligonucleotides denominated N15 and N16, which map at nucleotide positions 397-420 and 1417-1394, respectively, of the rat GnT II cDNA sequence (Fig. 3). The 3`-untranslated probe was generated by PCR amplification of a rat GnT II genomic clone fragment, using the T3 and T7 promoter primers. The human -actin cDNA probe was a 2.0-kilobase pair BamHI fragment derived from plasmid pHFA-1(32) , kindly donated by Peter Gunning. Hybridization with the human -actin cDNA probe was carried out at 37 °C, and membranes were washed at 50 °C.


RESULTS

Isolation of cDNA Clones for Rat GnT II

The GnT II enzyme was purified over 50,000-fold from rat liver homogenates. It was isolated in a quantity of approximately 100 µg in about 2.5% yield. The specific activity of the purified enzyme was 12 µmol of GlcNAc transferred/min/mg of protein. SDS-PAGE analysis of the purified enzyme under reducing conditions revealed a major polypeptide having a M of 42 kDa. N-terminal sequence analysis of the unreduced protein after reversed-phase HPLC yielded two sets of phenylthiohydantoins, in approximately equal amounts, for the first four cycles. However, by cycle five, a single major sequence of 33 residues became predominant, the other sequence apparently becoming less abundant (, experiment 1). Therefore, the protein was reduced and alkylated, preparatively run on SDS-PAGE, and electroblotted onto a polyvinylidene difluoride membrane. N-terminal sequence analysis of the 42-kDa GnT II polypeptide yielded two amino acid sequences (, experiment 2), which indicated, based on a partial overlap that could be discerned between the major and minor amino acid sequences, a ragged N terminus. Sequence analysis of a tryptic peptide derived from the purified 42-kDa GnT II polypeptide yielded a sequence of 21 residues (, experiment 3).

()Two 8-fold degenerate DNA oligomers of 17 nucleotides representing distinct segments of the amino acid sequence of the tryptic peptide of rat GnT II (underlined in ) were synthesized and used as hybridization probes to screen the oligo(dT)-primed rat liver cDNA library. The two oligonucleotide probes, having the sequences 5`-GTIGAYAARGAYGGIAC-3` and 5`-ARIACIARYTCICCIGG-3`, where R denotes A or G and Y denotes C or T, were designated P4 and P5, respectively. Approximately 6.2 10 recombinant phages were screened, using the DNA oligomer P4 as a hybridization probe. Seven clones, denominated R1, R3, R7, R9, R15, R17, and R20, were detected. Three clones, R1, R7, and R9, hybridized to both probes P4 and P5 and were isolated by plaque purification. A detailed restriction map was generated for each clone, and restriction fragments were subcloned in pBlueScript-based plasmid vectors for DNA sequence analysis. The clones R1, R7, and R9 contained cDNA inserts that were of similar length (1.9 kilobase pairs), showed a similar restriction pattern, and cross-hybridized (data not shown). The nucleotide sequence of the cDNA inserts from clones R7 and R9 was determined on both strands, as outlined in Fig. 1. The nucleotide sequence of the two cDNA clones was identical and is shown in Fig. 3, where it extends from nucleotide 268 to the terminal artificial EcoRI restriction site. Four additional cDNA clones, designated R9.2, R9.3, R9.4, and R9.5, were obtained through screening of the oligo(dT)-primed rat liver cDNA library, using the 826-bp EcoRI-HindIII fragment of clone R9 as a hybridization probe (Fig. 1). Restriction mapping and sequence analysis showed that none of these clones extend further in the 5`-direction than clones R7 and R9, their 5`-ends mapping to different regions within the nucleotide sequence of clones R7 and R9 (Fig. 1). The 3`-end of cDNA clones R9.2, R9.3, and R9.5 is identical to the 3`-end of cDNA clones R7 and R9, whereas the the 3`-end of cDNA clone R9.4 maps 115 bp upstream of the terminal EcoRI site. Although a canonical poly(A) addition signal (AATAAA) is located 16 bp upstream of the artificial EcoRI site in clones R7, R9, R9.2, R9.3, and R9.5, no poly(A) tract is present in these clones nor in clone R9.4. Therefore, these cDNA clones may represent self-priming events that occurred within the GnT II mRNA.

To isolate cDNA clones extending further in the 5`-direction than clones R7, R9, and R9.5, cDNA was synthesized from rat liver poly(A)-enriched RNA by priming reverse transcription with the GnT II-specific oligonucleotide R9P1R (Fig. 2). Following addition of a poly(C) tract to the 3`-end, the single-stranded cDNA was amplified by PCR using a nested GnT II-specific oligonucleotide designated R9P3R (Fig. 2) as a 3`-primer and an anchor oligonucleotide containing a poly(G) tract as a 5`-primer. Two major cDNA fragments of approximately 380 and 260 bp were detected in an agarose gel stained with ethidium bromide. No detectable DNA fragments were generated in a control reaction in which reverse transcriptase had been omitted (data not shown). To verify the authenticity of the two products of reverse transcription, the two cDNA fragments of 380 and 260 bp in length were separated by gel electrophoresis, and each fragment was further amplified by PCR using the adaptor oligonucleotide as a 5`-primer and either the GnT II-specific oligonucleotide R9P3R or R9P4R (Fig. 2) as a 3`-primer. As illustrated in Fig. 2, PCR amplification of each cDNA fragment using the two nested GnT II-specific oligonucleotides R9P3R and R9P4R generates two products that differ in size by 60 bp. This value closely matches the distance between the two oligonucleotides along the sequence of rat GnT II cDNA (Fig. 2), thus indicating that both cDNA species are reverse transcription products of rat liver GnT II mRNA, the smaller fragment probably being the result of pausing events during first-strand cDNA synthesis. Therefore, the larger cDNA fragment of 380 bp in length was cloned into a plasmid vector. The nucleotide sequence of the cDNA inserts of five recombinant plasmids, designated p5R7, p5R13, p5R16, p5R27, and p5R33, was determined on both strands, as outlined in Fig. 1. The sequences of all of these cDNA clones where they overlapped were identical and aligned perfectly to the sequence of the corresponding genomic region. The overlapping cDNA sequence of clones p5R7, p5R13, p5R27, R7, R9, R9.2, R9.3, R9.4, and R9.5 is shown in Fig. 3.

Sequence Analysis of Rat GnT II cDNA

The cDNA sequence predicts an open reading frame, which starts at the presumptive AUG translation initiation codon (nucleotide position 106) and continues for 1326 nucleotides to encode a polypeptide of 442 amino acids. The sequence context of this AUG is favorable for translation initiation. A purine is present at position -3. The presence of a purine at this position is the most critical and conserved feature that is observed in the flanking sequences of AUG codons utilized for initiation of protein synthesis in higher eukaryotes(33) .

The deduced amino acid sequence agrees with direct protein sequence data in 58 out of 59 residues (underscored with a solidbox in Fig. 3), which were positively identified by sequence analysis of the rat liver GnT II polypeptide (). The three amino acid sequences that were experimentally determined are encoded by distinct regions of the cloned cDNA, thus confirming that they represent different regions of a single translation product. At each one of these regions, the alignment between the deduced and experimentally determined amino acid sequence does not require the introduction of any gap, providing compelling evidence for colinearity. Inspection of the deduced amino acid sequence indicates that the two partially overlapping amino acid sequences observed in the sequence analysis of the purified 42-kDa GnT II polypeptide apparently represent the N-terminal amino acid sequences of two polypeptide species whose N termini differ in length by three amino acids, probably starting at residues Val-71 and Val-74. The N terminus of the tryptic peptide starts after a basic residue (Arg-100), as expected, and the unidentified amino acid residue at position 8 in the sequence of this peptide (, experiment 3) corresponds to a tryptophan residue (Trp-108), which can be chemically modified during electrophoresis(19) . The amino acid sequence that became predominant after the first four cycles of the Edman degradation of the intact GnT II enzyme, which was purified by reversed-phase HPLC (, experiment 1) actually corresponds to a segment of the C-terminal region of the GnT II translation product. Clearly, after reduction and SDS-PAGE, this peptide was missed on the 10% gels that were used, peptides of <10 kDa either running close behind or with the stacking buffer off the end of the gel. Amino acid residues representing the N terminus of the 42-kDa GnT II polypeptides were also detected in the first four cycles of the Edman degradation of the intact GnT II enzyme. As the 42-kDa GnT II polypeptide has two species differing in length at the N terminus by three amino acids, and as prolines, which tend to give lower yields during the Edman cleavage step, were present near its N terminus, the sequence of the small C-terminal peptide became predominant. Taken together, these data indicate that two polypeptides comprise the purified, active GnT II enzyme, either as a result of proteolysis during purification or as a result of normal proteolytic processing in the endomembrane system, or both. The small polypeptide of undetermined size, which encompasses the C-terminal 64 amino acid residues, interacts strongly, possibly through disulfide bridges, with the larger polypeptide of 42 kDa, which extends from residues 71 to near 378 of the initial translation product. Both polypeptides migrated together during reversed-phase HPLC under conditions that may be assumed to be rather denaturing.

The deduced amino acid sequence of the rat GnT II polypeptide chain contains two potential N-linked glycosylation sites having the consensus sequence Asn-Xaa-Ser/Thr at Asn-64 and Asn-81. N-Linked glycosylation at Asn-64-Asp-Ser can probably be excluded, since it has never been observed at Asn residues, which form part of a site where Xaa is a Pro or Asp residue(12) . N-Linked glycosylation could occur at Asn-81-Leu-Thr, as suggested by the observation that Asn-81 was left unidentified by amino acid sequence analysis of the 42-kDa GnT II polypeptide (). The calculated molecular mass of the unglycosylated form of this polypeptide, which includes Asn-81 ( and Fig. 3), is 36,276 daltons, 5.7 kDa lower than the molecular mass of 42 kDa determined by SDS-PAGE.

Computer-assisted analysis by the algorithm of Kyte and Doolittle (24) with a span setting of 9 revealed a hydrophobic stretch of 20 amino acids occurring at residues 10-29 (underlined in Fig. 3). This is the only hydrophobic region in the GnT II polypeptide sufficiently long enough to span the membrane lipid bilayer. The structural features of this N-terminal hydrophobic segment predict that it may represent a signal sequence that remains uncleaved and functions as a stop transfer signal during membrane insertion. The sequence that follows the hydrophobic core does not agree with the consensus sequence context for a signal peptidase recognition and cleavage site(34, 35) . Clusters of charged amino acid residues flank the two sides of this hydrophobic domain. Since the N-terminal side is more positive with respect to the C-terminal side ((C - N) = -3)(36) , a type II membrane topology is possible either by the positive inside rule (37) or the charge difference model(36, 38) .

The cloned cDNA includes 105 bp of 5`- and 721 of 3`-flanking sequences in addition to the 1326 bp of protein coding region. Within the 5`-untranslated region, no AUG codons 5` to the presumptive AUG initiator codon at position 106 are observed. The 5`-untranslated sequence has a G + C content of 76% and probably extends further upstream. Two transcription start sites, which map at 450 and 435 bases upstream of the presumptive AUG translation initiation codon, were detected by RNase protection experiments using restriction fragments of a rat GnT II genomic clone encompassing 1.1 kilobase pairs of 5`-flanking DNA sequences. Although at a low frequency, 5`-untranslated regions of 400-600 bases in length have been observed among eukaryotic mRNAs(39) . The 3`-untranslated region of rat GnT II mRNA contains three canonical polyadenylation signals, having the consensus sequence AATAAA (bold in Fig. 3). A cluster of AUUUA sequence motifs (40, 41) is found in the region between the first and second polyadenylation signals. In addition, multiple characteristic short sequence repeats, which have been implicated in the turnover of the apolipoprotein II mRNA(42) , are found dispersed along the region flanked by the first and third polyadenylation signals.

Expression of Rat GnT II cDNA in COS-7 Cells

The identity of the cloned cDNA for rat GnT II was unambiguously verified by expressing the cDNA segment coding for the C-terminal region of the GnT II polypeptide in COS cells. An indication that the substrate binding site and the catalytic domain of the GnT II enzyme are located in the C-terminal region of the GnT II polypeptide was provided by the observation that the membrane-solubilized form of rat GnT II, which is enzymatically active(13) , encompasses the C-terminal 373 amino acid residues ( and Fig. 3). The structures of the plasmids used for transfection are illustrated in Fig. 4. In these plasmids, the rat cDNA sequence coding for the C-terminal 389 amino acid residues of the GnT II polypeptide was linked in frame (pLJ268_R9) or out of frame (pLJ269_R9) to the human cDNA segment coding for the cleavable signal peptide sequence of the IL-2 receptor. Plasmid pLJ268_R9 carrying the rat GnT II cDNA in frame codes for a hybrid protein that will be acted upon by the signal peptidase, the mature polypeptide being transported across the endoplasmic reticulum, through the Golgi apparatus, and released into the extracellular fluid. This was intended to facilitate detection of GnT II activity above the endogenous level. Indeed, cultured mammalian cells, which lack GnT II activity, are not yet available. Conversely, plasmid pLJ269_R9 carrying the rat GnT II cDNA out of frame encodes a short fusion protein prematurely terminating 13 amino acid residues after the signal peptidase cleavage site and was used as a negative control.

Since the 3`-untranslated region of rat liver GnT II mRNA appears to have structural features that could negatively affect its stability or translational proficiency(40, 41, 42) , this region was removed from the rat liver GnT II cDNA and replaced in the expression plasmids with the 3`-untranslated region of the rat pre-proinsulin II gene, which provides an efficient polyadenylation signal(28) . Furthermore, the 5`-untranslated region of the rat pre-proinsulin II gene was placed between the long terminal repeats of the Rous sarcoma virus (which act as a strong promoter) and the cDNA for the cleavable signal sequence of the human IL-2 receptor to provide an additional potential stabilizing element to the messages encoded by the hybrid constructs.

COS-7 cells were transfected either with plasmid pLJ268_R9 or pLJ269_R9 by liposome-mediated uptake. Cells and culture media were collected at various times post-transfection and assayed for GnT II activity in the presence or absence of the acceptor substrate MGn(+F). Cells transfected with plasmid pLJ268_R9 showed at 48 h post-transfection a 10-fold increase in GnT II activity above the endogenous level of mock-transfected cells. No significant increase of GnT II activity above the endogenous level was observed in COS-7 cells transfected with plasmid pLJ269_R9 carrying the rat cDNA out of frame (data not shown). The results reported in demonstrate that COS-7 cells transfected with plasmid pLJ268_R9 synthesize and release into the culture medium a polypeptide endowed with GnT II activity. A consistent and progressive increase of GnT II activity was observed at 48 and 72 h post-transfection in the culture medium of COS cells transfected with plasmid pLJ268_R9 as compared with the negligible level of GnT II activity present in the culture medium of COS-7 cells transfected with plasmid pLJ269_R9. The enhancement of GnT II activity at 48 and 72 h post-transfection was 71- and 77-fold, respectively. Since the increase of GnT II activity in the culture medium of COS cells depends on the correct reading frame of the transfected rat cDNA, the possibility that it may result from activation events of the endogenous GnT II gene induced or mediated upon transfection by the rat cDNA or vector sequences can be excluded.

To positively identify the product of recombinant GnT II catalyis, enough product (225 nmol) was purified, as described under ``Experimental Procedures,'' to obtain a H-NMR spectrum, as shown in panelb of Fig. 5. Characteristic of the product of GnT II catalysis was the major downfield shift of the 1-6-linked mannose H-2 signal, as compared with the acceptor substrate, from = 3.972 to 4.107 upon substitution at the 2 position with a -GlcNAc residue(43) . In addition, the intensity of the signals representing the 1-2-linked GlcNAc H-1 protons (doublet, = 4.555, J = 8.3 Hz) and N-acetamido methyl protons (singlet, = 2.052) in the product were increased 2-fold as compared with the substrate.

Expression of GnT II mRNA

To examine the transcription product(s) of the rat GnT II gene, poly(A)-enriched preparations of RNA from rat liver, brain, thymus, and spleen were subjected to Northern blot analysis. Three GnT II-specific probes containing the 5`-end, the coding sequence, or the 3`-untranslated region of the cloned cDNA were used. The results are illustrated in Fig. 6, where the same amount (5 µg) of poly(A)-enriched RNA was used in each lane. A major transcript of 2.8 kb in length that hybridized to all three GnT II-specific probes was detected in these organs. In addition, two minor transcripts of 2.1 and 1.7 kb in length that hybridized only to probes containing the coding sequence and the 5`-untranslated region of GnT II cDNA were detected in all rat organs examined (Fig. 6, panelsA and B). The lack of hybridization of the two minor transcripts to the 3`-untranslated region probe (Fig. 6, panelC) indicates that they do not result from RNA degradation. As a matter of fact, a single 2.1-kb mRNA species was detected by probing the same blots at intermediate stringency conditions with a human -actin cDNA probe (data not shown). The weak hybridization signals, which correspond to an RNA species of 5.0 kb, comigrate with 28 S ribosomal RNA and could be the result of nonspecific hybridization. As illustrated in Fig. 6, the overall levels of GnT II mRNA vary in different rat organs, being higher in the thymus and spleen and progressively lower in the liver and brain. The relative abundance of the major and two minor transcripts showed no variation.


Figure 6: Northern blot analysis. 5-µg aliquots of poly(A)-enriched RNA from rat liver (L), brain (B), thymus (T), and spleen (S) were fractionated by electrophoresis through a denaturing 1.1% agarose gel. Three distinct fragments of rat GnT II cDNA were used as hybridization probes. PanelA, a 365-bp EcoRI fragment of plasmid p5R7 (Fig. 1) containing the 5`-end of the cDNA (nucleotide position 1-348 in Fig. 3); panelB, a 1021-bp cDNA fragment containing the coding region and extending from nucleotide 397 to 1417 of the rat GnT II cDNA sequence reported in Fig. 3; panelC, a 574-bp fragment of a rat GnT II genomic clone containing the terminal 448 nucleotides of the 3`-untranslated region of rat GnT II cDNA (nucleotide position 1699-2147 in Fig. 3) and 126 bp of 3`-flanking DNA sequence. The autoradiographs illus-trated in panelsA and B refer to distinct blots derived from different portions of a gel. The autoradiograph illustrated in panelC refers to a blot derived from a separate gel. The migration of the two ribosomal RNAs is indicated by the arrows on the leftside of the threepanels.




DISCUSSION

Two independent experimental data indicate that the cloned rat cDNA encodes the GnT II enzyme. First, the colinearity between the nucleotide sequence of the cloned cDNA and 58 non-overlapping amino acid residues, which were identified by sequence analysis of the rat GnT II polypeptide (), was consistent with the translation frame (Fig. 3). Second, expression in COS cells of the rat cDNA region coding for the C-terminal 389 amino acids of the GnT II polypeptide linked in frame to a human cDNA segment encoding the cleavable signal peptide sequence of the IL-2 receptor resulted in the synthesis and release into the culture medium of a polypeptide endowed with GnT II activity (). This was formally demonstrated by H-NMR spectroscopy of the product of in vitro catalysis by the enzyme (Fig. 5). The cDNA potentially codes for the membrane-bound form of the enzyme. It contains an open reading frame of 1326 nucleotides that encodes a polypeptide of 442 amino acids, with a calculated molecular mass for the unglycosylated polypeptide of 51,109 Da. In the absence of information on the molecular size and N-terminal amino acid sequence of the membrane-bound form of GnT II, the AUG codon at position 106 (Fig. 3) is assumed to function as a translation initiation codon.

Analysis of the deduced amino acid sequence of the rat GnT II protein predicts a functional domain architecture and type II transmembrane topology typical of the glycosyltransferases that have been cloned to date(21, 44, 45, 46, 47, 48, 49) . The membrane-bound form of rat GnT II is comprised of a short (9 amino acids) N-terminal cytoplasmic segment, a 20-amino acid hydrophobic stretch, which may act as a membrane anchor and stop-transfer signal, and a large C-terminal lumenal region where the substrate binding site and the catalytic domain are located. This conclusion is consistent with the amino acid sequence analysis of the purified form of rat GnT II and the results of the expression studies. A protease-sensitive segment of 50 amino acids (residues 30-80 in Fig. 3) enriched in proline, glycine, and hydrophilic residues appears to separate the catalytic portion of GnT II from the membrane-anchoring domain. As postulated for other glycosyltransferases(21, 44) , this hydrophilic region may represent a solvent-exposed region of the enzyme, which could act as a flexible arm, enabling the catalytic domain to move relative to the plane of the membrane.

The subcellular compartmentation of GnT II as well as any signal(s) involved in its localization to Golgi membranes remain to be elucidated. It has been thought that the GnT II enzyme resides in the medial Golgi compartment, since analysis of Golgi membrane fractions from Chinese hamster ovary cells showed that GnT II activity co-migrates with the GnT I and -mannosidase II activities(50) . However, recent immunocytochemical studies indicate that the subcellular distribution of both GnT I (51) and -mannosidase II (52), previously thought to be medial Golgi resident enzymes, is less topographically restricted and shows cell type-specific variations.

GenBank and SwissProt data base searches did not reveal significant sequence similarity to known DNA or protein sequences, including those of other previously cloned glycosyltransferases. Moreover, an extensive computer-assisted comparison did not reveal regions of significant sequence similarity to other mammalian N-acetylglucosaminyltransferases, including human (45) and rabbit (46) GnT I, human (47) and rat (48) GnT III, and rat and murine GnT V(49) , indicating that these enzymes are encoded by unrelated genes. This is especially surprising in the case of GnT I and GnT II, which both catalyze the formation of a GlcNAc1-2Man product, both utilize UDP-GlcNAc as a donor substrate, both require Mn ions for catalysis, and both are encoded by a gene not interrupted by introns(53) .()It is possible that distinct N-acetylglucosaminyltransferases may have related three-dimensional structures and that only a limited number of widely dispersed amino acids are conserved along the otherwise variable peptide sequence.

The sequence similarity between rat and human GnT II cDNA is 89% in the coding region (nucleotides 106-1434 in Fig. 3) and 86% in the 3`-untranslated region (nucleotides 1435-2147).() Moreover, two canonical polyadenylation sites whose relative positions correspond to the first and third polyadenylation sites in the 3`-untranslated region of rat GnT II cDNA (Fig. 3), have also been conserved in the cloned human GnT II cDNA. The conserved sequence similarity of the 3`-untranslated region across mammalian species suggests a conserved function of this region, probably in regulating GnT II mRNA stability or translational proficiency.

Multiple GnT II transcripts, which differ in their total abundance, were detected in rat liver, brain, thymus, and spleen by Northern blot analysis, using a coding region-specific probe (Fig. 6, panelB). The DNA sequences coding for GnT II are organized in a single exon not interrupted by introns both in the rat and human genome. Therefore, the occurrence of a transcript of 2.8 kb in length indicates that the cloned cDNA may not include the complete 3`-untranslated region of rat GnT II mRNA. Considering that transcription of the rat GnT II gene starts at 450 bases upstream from the AUG translation initiation codon at position 106 of the cDNA (Fig. 3), a transcript of 2.8 kb in length can be accounted for by a 1.1-kb 3`-untranslated region, which extends 0.4 kb further downstream of the 3`-end of the cloned rat GnT II cDNA. This conclusion is consistent with the observation that none out of the eight cDNA clones that were isolated contained a terminal poly(A) tract, probably representing self-priming events during cDNA synthesis due to the formation of local secondary structures in the mRNA. The variation of the overall level of GnT II mRNAs observed in rat organs could be the result of a transcriptional control mediated by tissue-specific transcription factors that specifically recognize and bind to sequence motifs, as suggested by sequence analysis of the 5`-untranslated region of the rat GnT II gene.

The origin and functional significance of the two minor GnT II transcripts of 2.1 and 1.7 kb in length remain to be elucidated. These transcripts do not hybridize to the probe containing the terminal portion of the 3`-untranslated region of rat GnT II cDNA (Fig. 6, panelC). Although the possibility that these mRNAs represent the transcription products of a similar but distinct gene or pseudo GnT II gene cannot be excluded, it is intriguing to speculate that the two minor GnT II transcripts of 2.1 and 1.7 kb may result from the differential utilization of distinct poly(A) addition sites during maturation of a primary transcript. According to our current knowledge of the function of the 3`-untranslated region of eukaryotic mRNAs(54, 55, 56) , the hypothesis can be extended to suggest that the differential utilization of the poly(A) addition sites likely mediated by specific factors may regulate expression of the GnT II gene by generating mRNAs species that differ in length and functional properties.

Characterization of the transcription unit of the GnT II gene will aid in elucidating the molecular mechanisms by which expression of the GnT II enzyme is regulated during development and differentiation or altered in human diseases. Studies toward the identification of the nature of the genetic mutation(s) that cause(s) a defect of GnT II activity in HEMPAS patients are in progress.

  
Table: Amino acid sequence analysis

Sequence data were obtained by N-terminal Edman degradation of the rat liver GnT II enzyme (Exp. 1), the 42-kDa GnT II polypeptide after purification by SDS-PAGE and electroblotting (Exp. 2), and a tryptic peptide of the purified 42-kDa polypeptide (Exp. 3). The amino acid assignments at each cycle of the Edman degradation are reported in the one letter code. Upper case letters indicate firm assignments, whereas lower case letters indicate tentative assignments or minor sequences. X denotes an unidentified amino acid. The sequence data used for the synthesis of degenerate oligonucleotide probes are underlined.


  
Table: GnT II expression

Expression of GnT II activity in the culture medium of COS-7 cells transfected with plasmids carrying the rat GnT II cDNA linked in frame or out of frame to a segment of human cDNA coding for the cleavable signal peptide sequence of the IL-2 receptor is shown.



FOOTNOTES

*
The initial part of this work was supported by a Terry Fox Special Initiative Grant from the National Cancer Institute of Canada (to J. Carver and H. S.) and by a grant from the Medical Research Council of Canada (to H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U21662.

§
Supported by the ENEA.

**
Supported by the Biomembrane Institute.

To whom correspondence should be addressed: Dr. Brad Bendiak: The Biomembrane Institute, 201 Elliott Ave. West, Seattle, WA 98119. Fax: 206-281-9893 or Dr. Giacomo A. D'Agostaro: Laboratory of Physics, CR ENEA, Casaccia, S. P. Anguillarese, 301, 00060 Rome, Italy. Fax: 39-30486559.

The abbreviations used are: GnT II, UDP-N-acetylglucosamine:-6-D-mannoside -1,2-N-acetylglucosaminyltransferase II; GnT I, UDP-N-acetylglucosamine:-3-D-mannoside -1,2-N-acetylglucosaminyltransferase I; GnT III, UDP-N-acetylglucosamine:-D-mannoside -1,4-N-acetylglucosaminyltransferase III; GnT V, UDP-N-acetylglucosamine:-6-D-mannoside -1,6-N-acetylglucosaminyltransferase V; GnT VI, UDP-N-acetylglucosamine:-6-D-mannoside -1,4-N-acetylglucosaminyltransferase VI; IL-2, interleukin 2; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; MGn(+F), Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-6)Glc-NAc; PCR, polymerase chain reaction; CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; TAPS, N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid; bp, base pair(s); kb, kilobase(s).

A. Zingoni, C. Petrarca, B. Bendiak, and G. A. F. D'Agostaro, manuscript in preparation.

J. Tan, G. A. F. D'Agostaro, B. Bendiak, F. Reck, J. Squire, P. Leong, and H. Schachter, submitted for publication.

G. A. F. D'Agostaro, F. Forbici, A. Zingoni, and B. Bendiak, manuscript in preparation.


ACKNOWLEDGEMENTS

We express our gratitude to Dr. Carla Marusic for advice in the sequence analysis of cDNA clones, to Roberto Biagini and Maria Cappelletti for technical assistance, and to Luigi Grisorio for artwork.


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