Oligosaccharyltransferase Isoforms that Contain Different Catalytic STT3 Subunits Have Distinct Enzymatic Properties  Daniel J. Kelleher, Denise Karaoglu, Elisabet C. Mandon, Reid Gilmore  Molecular Cell  Volume 12, Issue 1, Pages 101-111 (July 2003) DOI: 10.1016/S1097-2765(03)00243-0

Figure 1 Mammalian Homologs of Stt3p and Ost3p (A) The sequence identity between signal sequences (horizontal hatch), lumenal segments (gray), TM spans (black), and cytosolic loops (diagonal hatch) of human N33 and human IAP was calculated after alignment. (B) Predicted TM spans, cytosolic loops, and lumenal segments of human STT3-A and STT3-B are designated as in (A). Nonhomologous extensions and an insertion in the STT3-B sequence are indicated by altered hatching relative to STT3-A. (C) Peptide sequences used to elicit antisera. Labeled bars in (A) and (B) designate the location of peptides a–e. The expected specificity of antisera raised against the synthetic peptides is shown. Underlining (peptides a, b, and d) indicates nonconserved residues between a pair of homologs. Nonidentical residues in the human and mouse sequences are shown in lower case. Antigen c was a mixture of two peptides (one for IAP and one for N33-2). The underlined residues in peptide c are not present in N33-1 due to alternative splicing. (D) Digitonin extracts of canine microsomes (±Endo H digestion) were resolved by PAGE in SDS. Protein immunoblots were probed with antibodies to STT3-A or STT3-B. The asterisk designates a polypeptide that is recognized by the secondary antibody. Molecular Cell 2003 12, 101-111DOI: (10.1016/S1097-2765(03)00243-0)

Figure 2 Sedimentation of Detergent-Solubilized N33, IAP, STT3-A, and STT3-B (A) A digitonin-high-salt extract prepared from microsomes was resolved by glycerol gradient centrifugation, and aliquots from each of 14 fractions were assayed for OST activity. Fraction 1 is the top of the gradient. (B) Protein immunoblot analysis using antibodies specific for (a) ribophorin I, (b) STT3-A, (c) STT3-B, (d) IAP (e) N33 and (f) N33-2. Molecular Cell 2003 12, 101-111DOI: (10.1016/S1097-2765(03)00243-0)

Figure 3 Resolution of Canine OST Isoforms (A) The active glycerol gradient fractions (Figure 2) were resolved by Mono-Q ion exchange chromatography using a NaCl gradient in buffer A (see Experimental Procedures). Two of the activity peaks were pooled (pool A, fractions 13–15; pool C, fractions 18–21) for additional chromatography. (B) Rechromatography of pool A (squares) and pool C (circles) on separate Mono-Q columns using a chromatography protocol identical to (A). (C) Selected fractions from (B) were analyzed on a protein immunoblot to detect ribophorin I. The sample size for electrophoresis was adjusted based upon the OST activity shown in (B) as follows: pool A, 36 × 103 cpm/sample; pool B, 6 × 103 cpm/sample. (D) Resolution of pool A (squares) and pool C (circles) on separate Mono-Q columns using a NaCl gradient in buffer B. (E) Selected fractions from (D) were analyzed on a protein immunoblot to detect ribophorins I and II. The sample size for electrophoresis was adjusted based upon the OST activity shown in (D) as follows: pool A, 36 × 103 cpm/sample; pool C, fractions 20–23, 36 × 103 cpm/fraction; pool C, fractions 24–27, 6 × 103 cpm/fraction. The relative activity (OST activity/ribophorins) was calculated by dividing the OST activity (cpm of glycopeptide/gel lane) by the ribophorin content (R I + R II) as determined by densitometry of the protein immunoblot. (F) Samples of OST-III (1× and 8× loads) were resolved by PAGE in SDS and stained with Coomassie blue. Molecular Cell 2003 12, 101-111DOI: (10.1016/S1097-2765(03)00243-0)

Figure 4 OST Isoforms Differ in Subunit Composition (A) Pools B and C from a Mono-Q column similar to that shown in Figure 3A were combined for rechromatography as shown in Figure 3D. (B) Aliquots of fractions 15–29 from the Mono-Q column shown in (A) were analyzed on protein blots using antibodies specific for OST subunits (a–c, e) or Con A (d). Protein immunoblot e was probed with the antisera raised against synthetic peptide b to detect N33-1. The glycosylated OST subunits are labeled in blot d. Molecular Cell 2003 12, 101-111DOI: (10.1016/S1097-2765(03)00243-0)

Figure 5 Kinetic Properties of OST Isoforms (A) The activity of OST-I, OST-II, and OST-III was determined in assays that contained 5 μM Nα-Ac-Asn-[125I]Tyr-Thr-NH2 as the tripeptide acceptor and either Glc3Man9GlcNAc2-PP-Dol (G3; 700 nM or 1 μM), Man9GlcNAc2-PP-Dol (M9; 700 nM or 1 μM), or a donor substrate mixture (mix; 100 nM Glc3Man9GlcNAc2-PP-Dol plus 1 μM Man9GlcNAc2-PP-Dol). The rate of glycopeptide formation for the 700 nM and 1 μM assays were averaged to obtain apparent Vmax values, which are expressed as the turnover number. (B) The apparent Km for the tripeptide acceptor (Kp) was determined in assays that contained 5–90 μM tripeptide acceptor and either 600 nM Glc3Man9GlcNAc2-PP-Dol or 600 nM Man9GlcNAc2-PP-Dol. (C and D) Donor substrate saturation curves for assays that contained 2.1 nM OST-I or 9 nM OST-III were obtained for Glc3Man9GlcNAc2-PP-Dol (C) or Man9GlcNAc2-PP-Dol (D) in assays that contained 5 μM (C) or 10 μM (D) acceptor tripeptide. The experimental data were fit to Equation 1 to obtain values for Vmax (Vm), Ks, and αKs. The Kp values from (B) were used for curve fitting. (E) Glycopeptides from selected assays in (A) were resolved by HPLC to separate Glc3Man9GlcNAc2-NYT from Man9GlcNAc2-NYT. The HPLC profiles (offset for clarity) are from the following assays: (a) OST-II, 1 μM Glc3Man9GlcNAc2-PP-Dol; (b) OST-III, donor mix; (c) OST-II, donor mix; (d) OST-I, donor mix; (e) OST-II, 1 μM Man9GlcNAc2-PP-Dol. (F) The HPLC-resolved glycopeptides (E) were quantified to calculate the donor preference ratio (DP) using the following equation: DP = (M9-PP-Dol/G3-PP-Dol) × (G3-P/M9-P). M9-PP-Dol and G3-PP-Dol are the concentrations of the donor substrates, and G3-P and M9-P are the quantities, in cpm, of the glycopeptide products. Molecular Cell 2003 12, 101-111DOI: (10.1016/S1097-2765(03)00243-0)

Figure 6 Differential Expression of OST Subunits (A) Multiple tissue Northern blots were hybridized with probes specific for ribophorin I, STT3-A, STT3-B, IAP, N33, and β-actin. The hybridization positive bands are consistent with the expected mRNA sizes for ribophorin I (2.5 kb), STT3-A (2.7 kb), STT3-B (4.4 kb), IAP (2.2 kb), N33 (1.8 and 2.1 kb), and β-actin (2.0 kb). The β-actin mRNA probe served as a gel loading control. (B) Microsomes isolated from canine pancreas (CP), human fibroblasts (HF), and CH12.LX cells (CH12.LX) were resolved by PAGE in SDS and probed with antibodies to STT3-A or STT3-B. The amount of CP microsomes loaded per gel lane is expressed in eq (400 fmol OST/eq of CP microsomes). The quantity of HF and CH12.LX microsomes loaded per gel lane is derived from the indicated number of cells. Densitometry was used to estimate the amount of STT3-A and STT3-B in the HF and CH12.LX microsomes relative to the CP microsome standard. Molecular Cell 2003 12, 101-111DOI: (10.1016/S1097-2765(03)00243-0)

Figure 7 Evolutionary Tree of Eukaryotic STT3 Proteins The sequences of eukaryotic STT3 proteins were aligned with a ClustalW(1.4) program using the PAM-350 similarity matrix. The horizontal axis on the diagram is in changes per residue. The Genbank accession numbers for the sequences are shown. Molecular Cell 2003 12, 101-111DOI: (10.1016/S1097-2765(03)00243-0)