Glycans and cellular functions I - protein folding, ER processing, quality control

1. BCMB8130: Glycobiology Feb 13, 2007 • Glycans and cellular functions I - protein folding, ER processing, quality control Kelley Moremen http://bmbiris.bmb.uga.edu/moremen/BCMB8130Glycobiology/index.html • Complex Carbohydrate Research Center • 315 Riverbend Rd. • University of Georgia • Athens, GA 30602 • 706-542-1705 • moremen@uga.edu

2. Glc 2 Glc Glc Man Man Man 2 2 2 Man Man Man 2 3 6 Man Man 6 3 3 3 Man 4 GlcNAc 4 GlcNAc Asn Asn-linked glycosylation: what does it do and why do we care??? • Precursor structure conserved from yeast to mammals and plants (+ possibly archaebacteria). • Enormous cost in energy • Amazing conservation in linkage pattern and necessary genes/gene products (enzymes) • Standard dogma • Provides hydrophilicity (solubility) to the newly synthesized protein • Influences protein folding • Protection from proteases • Provides a scaffold for adding targeting information (i.e. lysosomal enzymes) • Scaffold for recognition by endogenous lectins (carbohydrate-receptor interactions) • Scaffold for exogenous recognition events (pathogen, toxin binding) BUT: Why start with this and only this structure?

3. Precursor synthesis The oligosaccharide is assembled sugar by sugar onto the carrier lipid dolichol High energy pyrophosphate bond http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

4. Helenius, A. and Aebi, M. (2004) Ann. Rev Biochem 73, 1019-49

5. Co-translational glycosylation is the rule from yeast to plants and animals. The steps in the synthesis of the lipid-linked precursor and the final structure are highly conserved. Even the exceptions are indicative of a highly conserved process: (From Alberts el al (1994) Molecular Biology of the Cell, 3rd ed.)

6. The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases Samuelson et al (2005) P.N.A.S. 102, 1548-1553 • The vast majority of eukaryotes synthesize Asn-linked glycans (Alg) by means of a LL precursor (Dol-PP-GlcNAc2Man9Glc3) • Characterized Alg glycosyltransferases and dolichol-PP-glycans of diverse protists.

7. Models for gain or loss of N-glycan biosynthesic enzymes Samuelson etal (2005) P.N.A.S. 102, 1548-1553 • Conclusions: • Common ancestry predicts the Alg glycosyltransferase inventory of each eukaryote. • This inventory accurately predicts the Dol-PP-glycans observed. • Alg glycosyltransferases are missing in sets from each organism. • Dol-PP-GlcNAc2Man5 and Dol-PP- and N-linked GlcNAc2 have not been identified previously in some of the wild type organisms. • Diversity of protist and fungal Dol-PP-linked glycans likely results from secondary loss of Alg genes from a common ancestor containing the complete set of Alg glycosyltransferases.

8. Transfer of the glycan to nascent polypeptide chains Higher eukaryotes: greatest efficiency for transfer of Glc3Man9GlcNAc2, but smaller glycans can be transferred under stress conditions (i.e. type 1 CDG patients where the LLO biosynthetic enzymes are compromised, mutant cell lines that are defective in LLO biosynthesis)

9. Structure of the Sec61 pore demonstrates the dimensions of co-translational extrusion of the polypeptide chain through the ER membrane van den Berg et al (2004) Nature 427, 36-44

10. van den Berg et al (2004) Nature 427, 36-44

11. Upon arrival in the ER Unfolded protein Native structure/ multimeric complex http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

12. Protein folding • Chaperones as general folding helpers: BiP prevents premature, incorrect folding of segments that arrive in the ER lumen • Formation of Disulfide bridges: ER is an oxidative environment which promotes the formation of SS bridges. Protein Disulfide Isomerase (PDI) to help with the formation of disulfide bonds or the rearrangement of disulfide bonds • PPI (peptide prolyl isomerase) to catalyze the isomerization of peptidyl-prolyl bonds, which can be rate-limiting in protein folding • Enzymes of the quality control cycle: calnexin (soluble) and caltreticulin (membranes associated) http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

13. Chaperones as folding helpers BiP (Hsp70 family member in the ER) keeps the newly arrived protein in an unfolded state to prevent premature folding of domains that have emerged earlier from the translocon Cleaved signal sequence http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

14. Protein disulfide isomerase (PDI) contains 2 cysteines that are close together and that are easily interconverted between the reduced SH form and the oxidized S-S form. NOTE: S-S bonds are only formed in the ER. Oxidized PDI facilitates the formation of SS bonds, acts as oxidant that is reduced. Reduced PDI helps with the formation of the correct disulfide bonds within the substrate (see figure). Exits unchanged from the reaction http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

15. Following protein folding in the ER E1 folding intermediates HS SH SH SH Semliki Forest virus infection of CHO cells SH S-S E1*; p62 (cleaved to generate E2) SH Pulse 1’; S-S S-S Ab: BiP Cnx Crt E1red Use of viruses: shut down host protein synthesis glycoproteins that transit ER “Tags” for following folding in ER: disulfide bonds glycosylation Eox1 Eox2 p62 has glycosylation site near N-terminus; E1 does not Molinari & Helenius (2000) Science 288: 331 http://www.biochem.wisc.edu/biochem702/ppts/lecture12.ppt

16. Benham & Braakman (2000) Crit Rev Biochem Mol. Biol. 35, 433-473

17. Peptidyl-prolyl-isomerase (PPI) PPI catalyzes the rotation about peptide prolyl-bonds, which can be rate limiting in the folding of protein domains http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

18. Protein Folding in the ER: Transport to the Golgi complex ER exit sites Classical ATP-driven Chaperones • Translation is initiated in the cytoplasm • Binding of SRP to the emerging polypeptide. • Ribosome-peptide-SRP complex binds to the SRP receptor on the ER membrane • Peptide is extruded through the Sec61 translocon in the ER membrane. • Glycans are added from Dol-P-OS to the emerging peptide via OST. BiP GRP94 GRP170 CRT PDI Lectin-based Chaperones SRP-Rec ERp57 OST BiP Dol-P-OS Sec61 CNX Ribosome SRP mRNA • Chaperones bind to facilitate protein folding: • ATP driven chaperones bind directly to polypeptides • Work in conjunction with PDI and PPIase to facilitate folding. • Lectin chaperones, CNX and CRT, bind to the glycan and facilitate folding in conjunction with ERp57, a PDI homolog. • Folded proteins are packaged at ER exits sites for transport to the Golgi complex

19. Carbohydrate structures influence glycoprotein folding in the ER -KDEL -KDEL Protein Folding (other chaperonins??) Calreticulin Glc II Glc II Glc Trans (Parodi enzyme) (only acts on unfolded glycoproteins) Glc I Glc II Anteriograde Transport to Golgi Glc II Glc II Endoplasmic Reticulum Protein Folding (other chaperonins??) Legend a1,2-Man P Calnexin OST a1,3-Man ER membrane P a1,6-Man Dol b1,4-Man b1,4-GlcNAc Cytoplasm a1,2-Glc a1,3-Glc

20. Events in the calnexin (CNX) cycle. Schrag et al (2003) TIBS 28, 49-57 • Membrane-anchored CNX recognize monoglucosylated high-mannose sugars attached to the unfolded or incompletely folded glycoprotein (red line). • Interactions with ERp57 facilitate the formation of proper disulfide bonds in the substrate glycoprotein. • Two possible tracks (blue or green arrows) upon release from CNX or calreticulin. • Folding and transport (green track) • Release of properly folded protein from CNX leads to deglucosylation of the terminal glucose by Glc II (ii). • ERGIC-53 recognizes the Man9GlcNAc2 glycoprotein for export out of the ER (iii). • Terminal misfolding -> disposal (blue track) • Terminal glucose of the glycan (pale blue) is removed by glucosidase II (Glc II) (ii). • Improperly or incompletely folded glycoproteins are reglucosylated (iii) by UGGT or a mannose residue (yellow) is trimmed by ER Man I (iv). • Additional cycles of binding by CNX or CRT follow reglucosylation by UGGT • Mannose trimming by mannosidase marks the protein for retrotranslocation and degradation.

21. Domain structures of calnexin and calreticulin. 3-D structure of calnexin calnexin • Lectin domain (blue) • Extended arm called the P-domain containing four repeat modules, P1–P4 (cyan, green, yellow and red). • Repeat modules formed by antiparallel interactions of two different proline-rich sequence motifs. • Glucose-binding site in the lectin domain is indicated by a ball-and-stick model (green) • Glc1Man9GlcNAc2 glycoprotein modeled by superimposing the terminal glucose on the observed position of bound glucose. • Position of the model glycoprotein (RNase B, magenta) suggests that interaction with the P-domain is likely. calreticulin glycan- binding protein-substrate binding ?? Schrag et al (2003) TIBS 28, 49-57

22. Helenius, A. and Aebi, M. (2004) Ann. Rev Biochem 73, 1019-49

23. Sequence alignment of lectin domains of CNX, ERGIC-53 and VIP-36 • Canine CNX and rat ERGIC-53 sequences were aligned according to the structural similarities (P-domains were omitted). • Pink boxes mark those residues whose C atoms are within 3 A° of their counterparts in the other structure. • Secondary structure assignments according to assignments in the Swiss-Pdb Viewer are shown (CNX in dark blue, ERGIC-53 in green). • Sequence identities of equivalent residues (those in pink boxes) are in bold purple characters. • Aligned residues that are farther apart than 3 A° are red. • Residues important in ligand binding in ERGIC-53 and their counterparts in VIP36 are yellow. • The alignment of the VIP36 amino acid sequence is based solely on visual comparison with that of ERGIC-53. Schrag et al (2003) TIBS 28, 49-57

24. Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system Pollock etal (2004) EMBO J. 23, 1020-1029 Calnexin and ERp57 act cooperatively to ensure a proper folding of proteins in the endoplasmic reticulum (ER). Calnexin contains two domains: a lectin domain and an extended arm termed the P-domain. ERp57 is a protein disulfide isomerase composed of four thioredoxin-like repeats and a short basic C-terminal tail. Here we show direct interactions between the tip of the calnexin P-domain and the ERp57 basic C-terminus by using NMR and a novel membrane yeast two-hybrid system (MYTHS) for mapping protein interactions of ER proteins. Our results prove that a small peptide derived from the P-domain is active in binding ERp57, and we determine the structure of the bound conformation of the P-domain peptide.

25. Recognition of local glycoprotein misfolding by the ER folding sensor UDP-glucose:glycoprotein glucosyltransferase Ritter and Helenius (2000) Nat. Structure Biol. 7, 278-280 UGGT acts as a folding sensor by specifically glucosylating N-linked glycans in misfolded glycoproteins thus retaining them in the calnexin/calreticulin chaperone cycle. To investigate how GT senses the folding status of glycoproteins, we generated RNase B heterodimers consisting of a folded and a misfolded domain. Only glycans linked to the misfolded domain were found to be glucosylated, indicating that the enzyme recognizes folding defects at the level of individual domains and only reglucosylates glycans directly attached to a misfolded domain. The result was confirmed with complexes of soybean agglutinin and misfolded thyroglobulin. BS: RNaseB subtilisin treated AS: RNaseA subtilisin treated Bsc: RnaseB with scrambled disulfides Asc: RnaseA with scrambled disulfides Solid bar: denstured domain Open bar: native domain

26. What happens if there are folding problems? glucosidases • Folding problem • Simply trapped in a malfolded conformation • mutation that leads to misfolding • unassembled multimer subunit glucosyltransferases Calnexin and BiP bind irreversibly to misfolded proteins Unfolded protein response pathway Increased transcription of chaperones and folding catalysts http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

27. Model for IRE1, XBP1, and ATF6 function in UPR Yoshida (2001) Cell 107, 881

28. UPR - Unfolded protein response (found in all eucaryotes) Response to many stimuli - anything affects protein folding in ER: affects glycosylation (glucose starvation, inhibitors of glycosylation such as tuni- camycin); ß-mercaptoethanol; calcium ionophores Induction of proteins during glucose starvation + glucose [3H] leucine labeling for 16 hours of mammalian (K12) cells; 2d gel electrophoresis; autoradiography Grp94 (hsp90 of ER lumen) Grp78 (hsp70 of ER lumen - BiP) hs) Hsc70 of cytosol Ac) actin • glucose Lee et al (1983) J. Biol. Chem. 259: 4616 http://www.biochem.wisc.edu/biochem702/ppts/lecture12.ppt

29. Failure of quality control -KDEL -KDEL Protein Folding (other chaperonins??) Calreticulin Glc II Glc II Glc Trans (Parodi enzyme) (only acts on unfolded glycoproteins) Glc I Glc II Anteriograde Transport to Golgi Glc II Glc II Endoplasmic Reticulum Protein Folding (other chaperonins??) Legend a1,2-Man P Calnexin OST a1,3-Man ER membrane P a1,6-Man Dol b1,4-Man b1,4-GlcNAc Cytoplasm a1,2-Glc a1,3-Glc

30. What happens when proteins can not fold correctly in the ER: failure in quality control Protein Folding (other chaperonins??) Calreticulin ER-associated Degradation (ERAD) Glc II Glc Trans (Parodi enzyme) (only unfolded glycoproteins) Glc I Glc II Translocation to cytoplasm through the Sec61 pore complex Glc II Endoplasmic Reticulum Protein Folding (other chaperonins??) P Calnexin OST ER membrane P Dol Proteasome Degradation Cytoplasm + Amino acids

31. What determines whether a protein is terminally misfolded? Glucose residues: determine interaction with calnexin/calreticulin The removal of a single mannose residue (by ER mannosidase I) while still in the cycle promotes association with EDEM (ER-degradation enhancing 1,2 mannosidase like protein) and targets the protein for ER associated degradation (ERAD). http://darwin.bio.uci.edu/~bardwell/231B_2005_Suetterlin_Lec2.ppt

32. Benham & Braakman (2000) Crit Rev Biochem Mol. Biol. 35, 433-473

33. Multiple steps in ER-associated degradation of unfolded glycoproteins (Jakob, C. A., Burda, P., Roth, J., and Aebi, M. (1998) J. Cell Biol. 142, 1223-1233) Protein Folding (other chaperonins??) Lectin? (EDEM?, Htm1p?) Step 2 ER Man I gene disruption Glc II Calreticulin Kif dMNJ Glc Trans (only unfolded glycoproteins) Glc I Glc II ER Man I Step 3 Step 1 Glc II Other Components? Endoplasmic Reticulum Protein Folding (other chaperonins??) Sec61 Translocon Calnexin P OST ER membrane P Dol Cytosolic N-glycanase Cytoplasm Proteosome Degradation Step 4 Two of the steps involve Class 1 mannosidases or mannosidase-related proteins

34. N-Linked oligosaccharide processing, but not association with CNX/CRT is highly correlated with ERAD of antithrombin Glu313-deleted mutant Tokunaga et al (2003) Arch. Biochem Biophys 411, 235-242 • Examined the combined effects of inhibitors of glycosidases, protein synthesis, proteasome, and tyrosine phosphatase on ERAD of a Glu313-deleted mutant of antithrombin. • Kifunensine suppressed ERAD (mannose trimming critical. • Cycloheximide and puromycin suppress ERAD, the effects cancelled by pretreatment with Cast. • Kifunensine suppresses ERAD even in Cast-treated cells, (does not require binding to CNX and/or CRT). • Inhibitors of ER mannosidase I and protein synthesis suppress ERAD at different stages and processing of N-linked oligosaccharides highly correlated with the efficiency of ERAD.

35. Overexpression of ERManI accelerates the timing clock for disposal (even for wild type proteins!!) • ERManI overexpression accelerates AAT-Z disposal (non-proteasomal->proteasomal) • ERManI overexpression targets WT AAT from fully secreted to fully degraded protein • ERManI acts is the rate-limiting timer for targeting proteins for disposal Wu et al (2003) P.N.A.S. 100, 8229-8234

36. Glycoprotein Biosynthesis Endoplasmic Reticulum Golgi Complex Endo a-Man A Golgi Man III/IIx(??) ER Man II GnTI C dMNJ Sw Kif dMNJ Kif Glc II GnTI Golgi Man II GnTII Golgi Man IA/IB B ER Man I Glc II P Glc I UGGT OST P Dol ER membrane Cytosol Failure of Quality Control Lysosome Glycosylasyaraginase Legend Chitobiase  1,2-Glc 1,2-Man Cytosolic -Man  1,3-Glc  1,3-Man B Proteosome degradation 1,2-GlcNAc  1,6-Man Lys -Man Lys 1,6- Man Lys -Man Amino Acids  1,4-Gal  1,4-Man  2,6-NeuNAc  1,4-GlcNAc Quality Control Glycoprotein Degradation (ERAD)

37. Structure of the yeast ER a-mannosidase I Howell and Herscovics labs (Vallee, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588) Side view of (aa)7 barrel End view of (aa)7 barrel

38. Structure of the yeast ER a-mannosidase I Howell and Herscovics labs (Vallee, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588) Protein unit 1 N-glycan 1 Protein unit 2 N-glycan2 Display of two protein units in the crystal lattice Active site in core of barrel

40. 1 2 3 4 6 5 6 7 8 11 9 10 12 13 14 14 Domain structures of Class 1 mannosidases EDEM1 EDEM2 EDEM3 KDEL ER MAN I Protease-associated domain Golgi Man IA

41. Threading of yeast HTM1 results in close fit with ER Man I Human ER Man I + DMJ Yeast HTM1 Overlap of side chains in catalytic core Ca backbone overlap

42. EDEM2 overexpression accelerates mutant 1antitrypsin disposal • EDEM2 overexpression can accelerate the degradation of AAT mutants PI Z or NHK • Acceleration of ERAD by EDEM2 requires a glycosylated ERAD substrate • Truncation of the COOH-terminal extension eliminates EDEM function in ERAD • EDEM2 in cell extracts can be found in a complex with calnexin by co-IP (data not shown) • Partially purified EDEM2 has been tested for cleavage of Man9-5GlcNAc2: no cleavage detected with any oligosaccharide structure Mast et al (2005) Glycobiology 15, 421-436 Steve Mast

43. Analogies for ERAD Sifers, R.N. (2003) Science 299, 1330-1331

44. Cellular response to unfolded proteins in the ER Only let correctly folded proteins out of the ER (Quality Control) Degrade the unfolded proteins before they accumulate in the ER (ER-associated degradation, ERAD)

46. 12 11 1 2 10 9 3 8 4 5 7 6 Two routes for exit from the ER: gating the exit of correctly folded proteins down the secretory pathway -KDEL Calreticulin Protein Folding Glc II to Golgi Complex Glc II Glc Trans (Parodi enzyme) (only unfolded glycoproteins) Glc I Glc II ER-associated degradation (ERAD) Glc II Endoplasmic Reticulum Timing? Recognition? P Calnexin OST ER membrane P Dol Proteasome Degradation Cytoplasm + Amino acids

47. Cholera Toxin Is Exported from Microsomes by the Sec61p Complex Schmitz et al (2000) J. Cell Biol. 148, 1203-1212 • Cholera toxin is transported from the plasma membrane to the ER • The A1 subunit (CTA1) is transferred to the cytosol. • Export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using microsomes loaded with CTA1. • Export of CTA1 from the microsomes was time- and adenosine triphosphate-dependent and required lumenal ER proteins. • By co-IP CTA1 was shown to be associated during export with the Sec61p complex • Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, thus export of CTA1 depended on translocation competent Sec61p complexes. • Export of CTA1 indicated insertion of the toxin into the Sec61p complex from the lumenal side. • Suggests that Sec61p complex–mediated protein export from the ER is not restricted to ER-associated protein degradation but is also use by bacterial toxins for entry into the cytosol of the target cell.

48. ER-Golgi Traffic Is a Prerequisite for Efficient ER Degradation Taxis, Vogel, and Wolf (2002) Mol. Biol. Cell 13, 1806-1818 • Efficient degradation of soluble malfolded proteins in yeast requires a fully competent early secretory pathway (i.e. mutant forms of sec12, sec23, sec18, ufe1, or sed5 all caused delays in degradation of CPY*). • Mutations in proteins essential for ER-Golgi protein traffic severely inhibit ER degradation of the model substrate CPY*. • ER localization of CPY* was found in WT cells, but no other specific organelle for ER degradation could be identified by electron microscopy studies. • CPY* is degraded in COPI coat mutants, but only a minor fraction of CPY* or some other proteinaceous factor that is required for degradation seems to enter the recycling pathway between ER and Golgi. • They propose that the disorganized structure of the ER and/or the mislocalization of Kar2p, observed in early secretory mutants, is responsible for the reduction in CPY* degradation. • Also, mutations in proteins directly involved in degradation of malfolded proteins (Der1p, Der3/Hrd1p, and Hrd3p) lead to morphological changes of the endoplasmic reticulum and the Golgi, escape of CPY* into the secretory pathway and a slower maturation rate of wild-type CPY.

49. Quality control in the endoplasmic reticulum protein factory Sitia and Braakman (2003) 18, 891-894

50. Gardinier lecture “Principles in Molecular and Cell Biology http://www.uiowa.edu/~c156201/PDFLecs/PDFlecs2004/Gardinier.12.1.04.pdf