Site-Specific N-Glycosylation Regulates the GPS Auto-Proteolysis of CD97

UvA-DARE (Digital Academic Repository)

Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function

Hsiao, C.-C.

Publication date 2015 Document Version Final published version

Link to publication

Citation for published version (APA): Hsiao, C-C. (2015). Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function.

General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)

Download date:02 Oct 2021 Site-specific N-glycosylation regulates the GPS 2 auto-proteolysis of CD97

FEBS Lett. 2009 Oct 6;583(19): 3285-90

N-glycosylation regulates the GPS auto-proteolysis

journal homepage: www.FEBSLetters.org

Site-speciﬁc N-glycosylation regulates the GPS auto-proteolysis of CD97

Cheng-Chih Hsiao a,b, Kai-Fong Cheng b, Hsin-Yi Chen b, Yi-Hua Chou b, Martin Stacey c, Gin-Wen Chang b, Hsi-Hsien Lin b,* a Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan b Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan c Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK article info abstract

Article history: Auto-proteolysis at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is a hallmark of Received 2 July 2009 adhesion-GPCRs. Although defects in GPS auto-proteolysis have been linked to genetic disorders, Revised 14 August 2009 information on its regulation remains elusive. Here, we investigated the GPS proteolysis of CD97, Accepted 1 September 2009 a human leukocyte-restricted and tumor-associated adhesion-GPCR. We found that CD97 is incom- Available online 6 September 2009 pletely processed, unlike its close homolog, epidermal growth factor-like module-containing Edited by Lukas Huber mucin-like hormone receptor 2. A unique pattern of N-glycosylation within the GPS motif of related adhesion-GPCRs was identified. The use of N-glycosylation inhibitors and mutants confirm site-specific N-glycosylation is an important determinant of GPS proteolysis in CD97. Our results suggest Keywords: Adhesion-G protein-coupled receptor that N-glycosylation may regulate the processing of adhesion-GPCRs leading to the production of Auto-proteolysis either cleaved or uncleaved molecules. G protein-coupled receptor proteolytic site Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. N-Glycosylation

1. Introduction Based upon the GRAFS phylogenetic classification system pro- posed by Fredriksson et al., the 33 adhesion-GPCR molecules con- Post-translational modification by proteolytic cleavage has stitute the second largest GPCR sub-family in the human proteome been recognized as an important mechanism for the functional [10]. The most distinct feature of adhesion-GPCRs is the chimeric modulation of a diverse array of cell surface proteins, such as composition of a long extracellular domain (ECD) and the seven- Notch, protease-activated receptors (PARs) and many cell adhesion span transmembrane (7TM) region [9,11]. Unusually large among molecules [1–3]. Proteolytic modification of these molecules usu- GPCRs, the ECD of adhesion-GPCRs typically consists of multiple ally takes place in the secretory pathway or on the cell surface, protein modules, a stalk region and a novel Cys-rich GPS domain. mediated by proprotein convertases or sheddases such as furin, Specifically, the N-terminus often contains common protein motifs MMPs and ADAMs [4]. known to be involved in protein–protein interactions, while the In addition to protease-mediated modifications, protease-inde- stalk region is rich in potential N- and O-glycosylation residues. pendent processing catalyzed by intramolecular cleavage (auto- The heavily glycosylated receptor was then dissected at the GPS proteolysis) has recently gained attention as an alternative mech- domain into extracellular a-subunit and 7TM b-subunit, which anism for receptor activation. Examples of these auto-proteolytic were then expressed on the membrane as a non-covalent hetero- modifications include the SEA module-mediated cleavage of dimer [9,11]. Muc-1 and dystroglycan [5–7], and the G protein-coupled receptor Our previous experiments on epidermal growth factor-like proteolytic site (GPS) domain-mediated cleavage of adhesion-G module-containing mucin-like hormone receptor 2 (EMR2), an epi- protein-coupled receptors (GPCRs) [8,9]. dermal growth factor module-containing seven-transmembrane receptor (EGF-TM7) member of adhesion-GPCR family, have shown that the GPS proteolysis is mediated by a self-catalytic reaction Abbreviations: 7TM, seven-transmembrane; DOC, degree of cleavage; ECD, extracellular domain; EGF-TM7, epidermal growth factor module-containing seven- similar to that of N-terminal nucleophile hydrolases [8]. We found transmembrane receptor; EMR2, epidermal growth factor-like module-containing that the GPS cleavage, which occurs in the endoplasmic reticulum mucin-like hormone receptor 2; Fc, fragment crystallisable; GPCR, G protein-coupled (ER), is critically dependent on the intact stalk region and is receptor; GPS, GPCR proteolytic site; PARs, protease-activated receptors; WT, wild- achieved via an intramolecular reaction involving the generation type and subsequent hydrolysis of a (thio)ester intermediate [8,12]. Ear- * Corresponding author. Fax: +886 32118700. E-mail address: [email protected] (H.-H. Lin). lier studies have suggested that proper cleavage at GPS might be a

35 prerequisite for efficient receptor trafficking to cell surface [13]. lyzed in 50 mM Tris–HCl, pH 7.4, buffer containing 10 mM CaCl2 More recently, deficiency in GPS cleavage has been linked to hu- and 150 mM NaCl at 4 °C. The purified proteins were subjected man genetic disorders. As such, point mutations affecting the to glycosidase treatment by incubation with 1 U of PNGase F, 1 U GPS cleavage of GPR56 and polycystin-1 have been implicated as of Neuraminidase (Roche Applied Science) in 20 mM sodium phos- the causes of bilateral frontoparietal polymicrogyria (BFPP) and phate buffer, pH 7.0, at 37 °C for 20 h prior to SDS–PAGE analysis. autosomal dominant polycystic kidney disease (ADPKD), respectively [14,15]. Therefore, GPS cleavage appears to be an inherent 2.4. Western blotting and other protein analysis and essential post-translational modification for the expression, presentation and function of adhesion-GPCRs. For Western blotting, proteins were separated in 8% or 10% Despite the direct link to diseases, little is known about the reg- SDS–PAGE gels, blotted and probed with anti-mFc or CLB/CD97-1 ulation of the novel self-catalytic GPS proteolytic reaction. In this mAbs. For the in vitro cleavage of mFc-fusion proteins, affinity- report, the GPS auto-proteolysis of CD97 was investigated. CD97 purified CD97-mFc, EMR2(125)-H516S-mFc, or EMR2(125)- is a leukocyte-restricted and tumor-associated EGF-TM7 molecule S518A-mFc-fusion proteins were incubated in cleavage buffer highly homologous to EMR2 [16,17]. The two molecules shared al- (50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA) with or without most identical EGF-like domains and 50% homology in the stalk 250 mM NH2OH (HA) at 37 °C. At various time points, samples and the 7TM regions. As well as functional roles in leukocyte were withdrawn and analyzed by Western blotting. For the quan- migration, recruitment and activation [18–21], CD97 is known to titative analysis of protein bands, the intensity of the uncleaved be involved in angiogenesis and the migration and invasion of tu- and cleaved protein bands on Western blots was quantified using mor cells [22–24]. By analyzing the GPS cleavage of CD97-mFc-fu- Gel-Pro Analyzer 3.1 (Media Cybernetics, MD, USA) according to sion protein, we show herein that CD97 and EMR2 have very the manufacturer’s instructions. different GPS cleavage efficiencies. Site-specific N-glycosylation in the stalk was found to play a critical role in regulating GPS auto-proteolysis of CD97. Our results suggest the adhesion-GPCRs 3. Results might adopt two different conformations, leading to the production of either cleaved or uncleaved molecules. 3.1. Detection of different GPS auto-proteolytic activities in CD97 and EMR2

2. Materials and methods While investigating the GPS cleavage of EMR2-mFc in previous experiments, CD97-mFc was included for comparison. Surpris- 2.1. Reagents and cell culture ingly, we detected very different degree of cleavage (DOC) in the two molecules; EMR2-mFc is almost entirely cleaved (>95% DOC) General chemicals were of analytical grade and obtained from as reported, but the cleavage of CD97-mFc is only partial ( 50% Sigma (Dorset, UK), unless otherwise stated. CLB/CD97-1 mAb DOC) (Fig. 1A). The same results were observed in all cell lines (mouse IgG2a) and EMR2-specific 2A1 mAb (mouse IgG1) were tested including CHO-K1, COS-7 and HEK-293T (data not shown). purchased from AbD Serotec (Kidlington, UK). Horseradish peroxi- Most importantly, similar results were seen in the membrane form dase (HRP)-conjugated 2nd Ab was purchased from Sigma. All cul- of CD97, indicating that the cleavage of CD97-mFc mimics that of ture media were from Invitrogen (Carlsbad, CA) and were CD97 receptor (Supplementary Fig. S1). A point mutation at the supplemented with 10% heat inactivated fetal calf serum (FCS), GPS site (CD97-S531A) completely inhibits the cleavage, suggest- 2 mM L-glutamine, 50 IU/ml penicillin and 50 lg/ml streptomycin. ing the cleavage mechanism of CD97 is highly similar, if not iden- HEK-293T and COS-7 were cultured in Dulbecco’s Modified Eagle tical, to that of EMR2 (Supplementary Fig. S1). To further confirm Medium (DMEM). CHO-K1 cells were grown in Ham’s F-12 med- the partial cleavage of CD97-mFc, fusion proteins were affinity ium. Lec1 cells were maintained in a-minimum essential medium purified and analyzed by SDS–PAGE. As shown in Fig. 1B, two (a-MEM). Transient transfection of expression constructs by Lipo- broad bands of 95–125 kDa and 50–75 kDa were identified, TM fectamine (Invitrogen) was performed as previously described representing the unprocessed molecule and the cleaved extracellu- [8]. For the use of N-glycosylation inhibitors, cells were incubated lar subunit, respectively. A third distinct band of 35 kDa is that of with the following inhibitors: tunicamycin at 0.1 lg/ml, 1 lg/ml cleaved mFc fragment. Subsequent de-glycosylation reaction con- and 10 lg/ml, castenospermine (CAS) at 50 lg/ml, 1-deoxynojiri- firmed the two broad bands are indeed due to heavy glycosylation mycin (DNJ) at 100 mM, swainsonine (SW) at 10 lg/ml. In all cases, as reported (Fig. 1B and Supplementary Fig. S2) [26]. cells were pre-treated with the inhibitors for 12 h, replaced with We have previously shown that strong nucleophiles such as fresh OPTI-MEM I medium containing inhibitors for a further 24 h. hydroxylamine (HA) can greatly enhance the processing rate of an incomplete GPS proteolytic reaction [8]. This is achieved by 2.2. Construction of expression vectors facilitating the hydrolysis of the (thio)ester intermediate, the rate-limiting step of the GPS proteolytic reaction. To investigate All expression vectors were constructed using pcDNA3.1(+) or whether the uncleaved CD97-mFc identified here is the result of pcDNA3.1/myc-His vectors (Invitrogen) unless otherwise specified. an incomplete proteolytic reaction (i.e. a stalled intermediate) or EMR2- and CD97-mFc expression constructs have been described a truly unprocessed product, purified proteins were treated with previously [8,25]. The chimeric and N-glycosylation site mutants HA in an in vitro cleavage reaction (Fig. 1C). As expected, enhanced were generated using polymerase chain reaction (PCR) amplifica- cleavage was observed in a ‘‘cleavage-incomplete” protein, EMR2 Ò tion by Advantage HF 2 PCR Kit (BD) using site-specific mutant H516S-mFc, in the presence of HA. On the contrary, no cleavage primers (Supplementary Table S1). was detected in cleavage-deficient EMR2 S518A-mFc. Similar to the cleavage-deficient EMR2 molecule, CD97-mFc showed no en- 2.3. Production, purification and glycosidase treatment of mFc-fusion hanced proteolysis upon HA treatment. This indicated that the proteins uncleaved CD97-mFc is a cleavage-incompetent molecule (Fig. 1C). Altogether, these results suggest that CD97-mFc can The production and purification of mFc-fusion proteins were adopt two different conformations; one capable of auto-proteolysis carried out as described previously [8]. Purified proteins were dia- and the other not.

36 N-glycosylation regulates the GPS auto-proteolysis

A BC 2

Fig. 1. Detection of different GPS auto-proteolytic activities in CD97 and EMR2. (A) The EGF-like modules and the stalk region are represented by triangles and a black line, respectively. The GPS cleavage site is indicated by an arrow. Western blot analysis of the CD97-mFc (lane 4) and EMR2-mFc (lane 3) is shown. Lanes 1 and 2 are mock and mFc-only control, respectively. Blots were probed with HRP-conjugated anti-mouse Fc-specific mAb that detects the uncleaved protein ( 100 kDa) and the mFc subunit ( 36 kDa). (B) Affinity purified CD97-mFc proteins were digested without (lane 1) or with PNGase and neuraminidase (lane 2) and visualized by Coomassie brilliant blue staining. M represents the protein marker (in kDa). The uncleaved protein, the cleaved ECD and mFc subunits are represented by black arrowhead, white arrowhead and asterisk, respectively. (C) Western blot analysis of CD97-mFc and mutant EMR2-mFc-fusion proteins subjected to in vitro cleavage reaction in the presence of NH2OH (HA) at 37 °C as indicated.

A C

Fig. 2. GPS auto-proteolysis is mainly regulated by the stalk region. (A) Diagrams depict the EMR2-mFc, CD97-mFc and domain-swapping fusion proteins (indicated by numbers 1–6, respectively). Western blot analysis of these fusion proteins is shown in the right panel. (B) Sequence alignments of the GPS motifs that are known to be cleaved. The conserved residues are highlighted in grey background. The arrow and the boxes indicate the cleavage site and the potential N-glycosylation sites, respectively. The abbreviations represent animal species (h, human; m, mouse; r, rat; d, Drosophila). (C) Western blot analysis of cleavage of the mFc-fusion proteins. Samples include mock-transfected cells (lane 1), cells transfected with constructs expressing mFc (lane 2), EMR2-mFc (lane 3), CD97-mFc (lane 4), EMR3-mFc (lane 5) and EMR4-mFc (lane 6) proteins.

3.2. Auto-proteolysis of CD97-mFc is regulated predominantly by the (also see Supplementary Fig. S3A). A closer examination of the stalk stalk region region of related adhesion-GPCRs identiﬁed an interesting pattern of potential N-glycosylation sites. A minimum of one N-glycosyla- To locate the region involved in the regulation of GPS auto- tion site is present in the GPS motif of all adhesion-GPCRs sur- proteolysis, we established and compared domain-swapping veyed, except EMR2 (Fig. 2B). Furthermore, 50% and 75% DOC chimeras of EMR2-mFc and CD97-mFc because of their strong sim- was identiﬁed in EMR3 and EMR4, two EGF-TM7 members that ilarity (Fig. 2). As expected, Fig. 2A shows the auto-proteolytic contain two and one N-glycosylation sites in the GPS motif, respec- activity is mainly determined by the less homologous stalk region tively (Fig. 2C and Supplementary Fig. S3B) [27,28]. These results

37 suggest that N-glycosylation in the stalk region (and the GPS 3.4. The role of site-specific N-glycosylation in the regulation of GPS domain) might play a role in determining the conformation of proteolysis in CD97-mFc EGF-TM7-mFc-fusion proteins and hence the DOC. A total of 5 potential N-glycosylation (G) sites were identified in 3.3. Regulation of GPS proteolysis in CD97-mFc by N-glycosylation the stalk regions of CD97 (Fig. 4A). To investigate the role of site- specific N-glycosylation in GPS auto-proteolysis, we created and N-Linked glycans play an essential role in ER quality control as a evaluated the DOC of single and multi-N-glycosylation site mu- vital determinant for the proper folding of proteins. To examine the tants of CD97-mFc proteins. In comparison to the wild-type (WT) role of N-glycosylation in regulating GPS cleavage, CD97-mFc and protein, all single N-glycosylation site mutants showed slight ef- EMR2-mFc transfected cells were treated with various N-glycosyl- fects on the DOC either positively or negatively (Fig. 4B and Sup- ation inhibitors (Fig. 3A and B). Tunicamycin interferes with the plementary Fig. S4A). To show this effect is specific and not first step of glycoprotein synthesis by inhibiting the enzymatic simply due to residue changes, we established single mutants reaction catalyzing the transfer of core oligosaccharides to the nas- replacing the three individual residues of the first N-glycosylation cent peptide chain. Because N-glycosylation influenced protein site (G1) (N371Q, V372A and T373A). The DOC of V372A mutant, secretion, we could not detect CD97-mFc and EMR2-mFc proteins which should preserve N-glycosylation, was similar to the WT pro- in the conditioned media of tunicamycin-treated cells. However, tein. On the other hand, both N371Q and T373A mutants all we noticed the DOC of newly synthesized proteins within the showed similar enhanced DOC (Fig. 4B). tunicamycin-treated cells was greatly reduced (Fig. 3A). The reduc- Intriguingly, significant modulation of GPS proteolysis was ob- tion is observed for both CD97-mFc and EMR2-mFc, but is more served in multi-N glycosylation site mutants. The cleavage of prominent in CD97-mFc, rendering it almost entirely uncleaved. CD97(G2,3), CD97(G1-3), CD97(G2-4), CD97(G2-5), and CD97(G1- Unlike the tunicamycin treatment however, no significant effect 5) mutants was greatly reduced (only 1–10% DOC), whereas the on the DOC of CD97-mFc and EMR2-mFc was observed in cells processing of CD97(G4,5) and CD97 (G1,4,5) mutants was effi- treated with castanospermine (CAS) and 1-deoxynojirimycin ciently enhanced with the DOC of CD97(G4,5) reaching 95% (DNJ), both glucosidases I and II inhibitors (Fig. 3B). The same is (Fig. 4B and Supplementary Fig. S4B). These results indicate that true for cells treated with swainsonine (SW), a Golgi alpha-man- site-specific N-glycosylation in the stalk region is critically in- nosidase II inhibitor. Likewise, when analyzed in Lec1 cells, which volved in the GPS auto-proteolysis of CD97-mFc. Specifically, com- nd lacks N-acetylglucosaminyltransferase I activity, the DOC of both bined mutation at the 2 and 3rd glycosylation sites, G2(N406) and EMR2-mFc and CD97-mFc was not altered (Fig. 3C). These results G3(N413), seem to reduce GPS auto-proteolysis. Conversely, muta- are consistent with the consensus that proteolysis takes place tion involving the 4th and 5th sites (G4,5) greatly enhances prote- within ER. Our data further indicate that GPS cleavage occurs after olysis. Interestingly, the effect of G2,3 mutations on the DOC seems the attachment of the oligosaccharide precursor, but does not de- to dominate over that of G4,5 mutations, as exemplified by the pend on its subsequent processing. mostly uncleaved CD97(G2-5) and CD97(G1-5) mutants (Fig. 4B).

Fig. 3. Regulation of GPS proteolysis of CD97-mFc molecule by N-glycosylation. (A) Western blot analysis of the effect of tunicamycin on the GPS cleavage of CD97-mFc and EMR2-mFc proteins. (B) Western blot analysis of the effect of various N-glycosylation inhibitors (CAS, DNJ, SW) on the GPS cleavage of CD97-mFc and EMR2-mFc proteins. (C) The expression patterns of CD97-mFc (lane 2) and EMR2-mFc (lane 1) in Lec 1 cells.

38 N-glycosylation regulates the GPS auto-proteolysis

A B 2

Fig. 4. Regulation of GPS proteolysis of CD97-mFc molecule by site-speciﬁc N-glycosylation. (A) Diagram depicting the wild-type (WT) CD97-mFc molecule, with a special attention on the potential N-glycosylation sites. The 5N-glycosylation sites in the stalk were denoted as G1, G2, G3, G4, and G5, respectively. (B) Western blot analysis of various CD97-mFc proteins including the WT and N-glycosylation site mutants.

4. Discussion (Figs. 2–4). Consistent with the consensus that GPS cleavage takes place in the ER lumen, we hypothesize that N-glycosylation at spe- As evidenced by genetic diseases caused by mutations that af- cific sites of CD97 stalk promotes the folding of CD97-mFc precur- fect GPS cleavage, the self-catalytic proteolysis at the GPS domain sor to a favorable conformation to allow GPS cleavage reaction. If is no doubt an essential post-translational modification of the GPS the favorable condition is not met due to differential N-glycosyla- domain-containing receptors. However, several recent studies tion, the precursor molecule will adopt a different conformation have reported the detection of both cleaved and uncleaved adhe- that is cleavage-incompetent and thus is expressed as an unc- sion-GPCRs, including GPR56 and flamingo, in vivo [29,30]. These leaved single-chain protein. results strongly suggest that the GPS auto-proteolytic reaction is Because of the heterogenous nature of the N-glycosylation site regulated and that the cleaved and uncleaved adhesion-GPCRs occupancy [33], it is possible to produce receptors containing var- could potentially have different cellular functions. This is consis- iable degrees of N-glycosylation leading to cleavable and uncleav- tent with the recent finding by Yu et al. on the functions of polycy- able conformations. This implies a regulatory mechanism whereby stin-1, a GPS motif-containing 11-TM molecule associated with the eventual cellular function of a specific adhesion-GPCR could ADPKD [31]. By comparing the phenotypes of the Pkd1 null and a possibly be determined by the efficiency of site-specific N-glyco- specific Pkd1 knock-in animal model expressing only the noncleav- sylation, and hence the relative amounts of the cleaved and unc- able polycystin-1, Yu and colleagues demonstrated that the leaved receptors. From this point of view, it is interesting to note cleaved polycystin-1 is critical for kidney tubular integrity, while that all GPS domain-containing receptors, including adhesion- the uncleaved polycystin-1 is required for embryonic development GPCRs and polycystin-1, contain an extracellular stalk rich in po- [31]. The fact that both cleaved and uncleaved polycystin-1 mole- tential N- and O-glycosylation sites that are in close proximity of cules have distinct biological functions is also reflected by the the GPS motif. It will be interesting in the future to analyze expression of the respective protein products in vivo [31,32]. whether different factors other than N-glycosylation are involved These observations indicated that although the GPS proteolysis in the regulation of GPS auto-proteolytic reaction. Such informa- is self-catalytic, the reaction is probably not 100% efficient. Alter- tion should in turn help shed lights on the generation and the bio- natively, it is possible that the adhesion-GPCRs can adopt different logical functions of the cleaved and uncleaved adhesion-GPCRs. conformations and proceed with or without GPS cleavage. Our data on the regulation of GPS cleavage in CD97 seem to support the lat- Acknowledgements ter mechanism. Likewise, a similar conclusion was reached by Wei et al. who demonstrated that endogenous polycystin-1 can mature We thank Dr. J.Q. Davies for his critical comments. This work was via two different pathways leading to the production of uncleaved supported by grants from National Science Council (NSC96-2320-B- full-length molecule as well as cleaved products [32]. The degree of 182-005) and Chang Gung Memorial Hospital (CMRPD170012) to cleavage (DOC) shown in this report was determined by comparing H.-H. Lin. the ratio of the uncleaved to cleaved protein bands on Western blots. This was based upon the assumption that all mutant proteins Appendix A. Supplementary data have similar protein stability. As N-glycans are important for protein stability, it should be cautioned that it is also likely that the re- Supplementary data associated with this article can be found, in moval of N-glycans at certain positions can influence the overall the online version, at doi:10.1016/j.febslet.2009.09.001. protein stability and turn-over. This effect, if occurred, will affect the measurement of DOC, even though it might not really affect References GPS cleavage. Detailed structural analysis of these individual proteins by other means such as the circular dichroism spectroscopy [1] Arora, P., Ricks, T.K. and Trejo, J. (2007) Protease-activated receptor signalling, or conformational unfolding studies in the future should help dis- endocytic sorting and dysregulation in cancer. J. Cell Sci. 120, 921–928. [2] Garton, K.J., Gough, P.J. and Raines, E.W. (2006) Emerging roles for ectodomain tinguish these potential effects. shedding in the regulation of inflammatory responses. J. Leukoc. Biol. 79, Based upon available experimental evidence, we suggest a 1105–1116. model where the folding of adhesion-GPCRs in ER can lead to [3] Higashiyama, S., Iwabuki, H., Morimoto, C., Hieda, M., Inoue, H. and either a cleavable or uncleavable conformation. In CD97-mFc, the Matsushita, N. (2008) Membrane-anchored growth factors, the epidermal growth factor family: beyond receptor ligands. Cancer Sci. 99, 214–220. structural determinant for the two distinct conformations was [4] Page-McCaw, A., Ewald, A.J. and Werb, Z. (2007) Matrix metalloproteinases found to include site-specific N-glycosylation in the stalk region and the regulation of tissue remodelling. Nat. Rev. Mol. Cell Biol. 8, 221–233.

39 [5] Akhavan, A., Crivelli, S.N., Singh, M., Lingappa, V.R. and Muschler, J.L. (2008) [20] Veninga, H. et al. (2008) Analysis of CD97 expression and manipulation: SEA domain proteolysis determines the functional composition of antibody treatment but not gene targeting curtails granulocyte migration. J. dystroglycan. FASEB J. 22, 612–621. Immunol. 181, 6574–6583. [6] Levitin, F. et al. (2005) The MUC1 SEA module is a self-cleaving domain. J. Biol. [21] Wang, T. et al. (2007) Improved antibacterial host defense and altered Chem. 280, 33374–33386. peripheral granulocyte homeostasis in mice lacking the adhesion class G [7] Macao, B., Johansson, D.G., Hansson, G.C. and Hard, T. (2006) Autoproteolysis protein receptor CD97. Infect. Immun. 75, 1144–1153. coupled to protein folding in the SEA domain of the membrane-bound MUC1 [22] Aust, G., Steinert, M., Schutz, A., Boltze, C., Wahlbuhl, M., Hamann, J. and mucin. Nat. Struct. Mol. Biol. 13, 71–76. Wobus, M. (2002) CD97, but not its closely related EGF-TM7 family member [8] Lin, H.H., Chang, G.W., Davies, J.Q., Stacey, M., Harris, J. and Gordon, S. (2004) EMR2, is expressed on gastric, pancreatic, and esophageal carcinomas. Am. J. Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein- Clin. Pathol. 118, 699–707. coupled receptor proteolytic site motif. J. Biol. Chem. 279, 31823–31832. [23] Galle, J., Sittig, D., Hanisch, I., Wobus, M., Wandel, E., Loeffler, M. and Aust, G. [9] Yona, S., Lin, H.H., Siu, W.O., Gordon, S. and Stacey, M. (2008) Adhesion-GPCRs: (2006) Individual cell-based models of tumor–environment interactions: emerging roles for novel receptors. Trends Biochem. Sci. 33, 491–500. multiple effects of CD97 on tumor invasion. Am J Pathol 169, 1802–1811. [10] Fredriksson, R., Lagerstrom, M.C., Lundin, L.G. and Schioth, H.B. (2003) The G- [24] Wang, T., Ward, Y., Tian, L., Lake, R., Guedez, L., Stetler-Stevenson, W.G. and protein-coupled receptors in the human genome form five main families. Kelly, K. (2005) CD97, an adhesion receptor on inflammatory cells, stimulates Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, angiogenesis through binding integrin counterreceptors on endothelial cells. 1256–1272. Blood 105, 2836–2844. [11] Stacey, M., Lin, H.H., Gordon, S. and McKnight, A.J. (2000) LNB-TM7, a group of [25] Stacey, M., Chang, G.W., Davies, J.Q., Kwakkenbos, M.J., Sanderson, R.D., seven-transmembrane proteins related to family-B G-protein-coupled Hamann, J., Gordon, S. and Lin, H.H. (2003) The epidermal growth factor-like receptors. Trends Biochem. Sci. 25, 284–289. domains of the human EMR2 receptor mediate cell attachment through [12] Chang, G.W., Stacey, M., Kwakkenbos, M.J., Hamann, J., Gordon, S. and Lin, H.H. chondroitin sulfate glycosaminoglycans. Blood 102, 2916–2924. (2003) Proteolytic cleavage of the EMR2 receptor requires both the [26] Chang, G.W. et al. (2007) CD312, the human adhesion-GPCR EMR2, is extracellular stalk and the GPS motif. FEBS Lett. 547, 145–150. differentially expressed during differentiation, maturation, and activation of [13] Krasnoperov, V., Lu, Y., Buryanovsky, L., Neubert, T.A., Ichtchenko, K. and myeloid cells. Biochem. Biophys. Res. Commun. 353, 133–138. Petrenko, A.G. (2002) Post-translational proteolytic processing of the calcium- [27] Stacey, M., Chang, G.W., Sanos, S.L., Chittenden, L.R., Stubbs, L., Gordon, S. and independent receptor of alpha-latrotoxin (CIRL), a natural chimera of the cell Lin, H.H. (2002) EMR4, a novel epidermal growth factor (EGF)-TM7 molecule adhesion protein and the G protein-coupled receptor. Role of the G protein- up-regulated in activated mouse macrophages, binds to a putative cellular coupled receptor proteolysis site (GPS) motif. J. Biol. Chem. 277, 46518– ligand on B lymphoma cell line A20. J. Biol. Chem. 277, 29283–29293. 46526. [28] Stacey, M., Lin, H.H., Hilyard, K.L., Gordon, S. and McKnight, A.J. (2001) Human [14] Piao, X. et al. (2004) G protein-coupled receptor-dependent development of epidermal growth factor (EGF) module-containing mucin-like hormone human frontal cortex. Science 303, 2033–2036. receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand [15] Qian, F. et al. (2002) Cleavage of polycystin-1 requires the receptor for egg on human macrophages and activated neutrophils. J. Biol. Chem. 276, 18863– jelly domain and is disrupted by human autosomal-dominant polycystic 18870. kidney disease 1-associated mutations. Proc. Natl. Acad. Sci. USA 99, 16981– [29] Iguchi, T., Sakata, K., Yoshizaki, K., Tago, K., Mizuno, N. and Itoh, H. (2008) 16986. Orphan G protein-coupled receptor GPR56 regulates neural progenitor cell [16] Gray, J.X. et al. (1996) CD97 is a processed, seven-transmembrane, migration via a G alpha 12/13 and Rho pathway. J. Biol. Chem. 283, 14469– heterodimeric receptor associated with inflammation. J. Immunol. 157, 14478. 5438–5447. [30] Usui, T., Shima, Y., Shimada, Y., Hirano, S., Burgess, R.W., Schwarz, T.L., [17] Hamann, J. et al. (1995) Expression cloning and chromosomal mapping of the Takeichi, M. and Uemura, T. (1999) Flamingo, a seven-pass transmembrane leukocyte activation antigen CD97, a new seven-span transmembrane cadherin, regulates planar cell polarity under the control of Frizzled. Cell 98, molecule of the secretion receptor superfamily with an unusual 585–595. extracellular domain. J. Immunol. 155, 1942–1950. [31] Yu, S. et al. (2007) Essential role of cleavage of Polycystin-1 at G protein- [18] Capasso, M., Durrant, L.G., Stacey, M., Gordon, S., Ramage, J. and Spendlove, I. coupled receptor proteolytic site for kidney tubular structure. Proc. Natl. Acad. (2006) Costimulation via CD55 on human CD4+ T cells mediated by CD97. J. Sci. USA 104, 18688–18693. Immunol. 177, 1070–1077. [32] Wei, W., Hackmann, K., Xu, H., Germino, G. and Qian, F. (2007) [19] Leemans, J.C., te Velde, A.A., Florquin, S., Bennink, R.J., de Bruin, K., van Lier, Characterization of cis-autoproteolysis of polycystin-1, the product of R.A., van der Poll, T. and Hamann, J. (2004) The epidermal growth factor-seven human polycystic kidney disease 1 gene. J. Biol. Chem. 282, 21729–21737. transmembrane (EGF-TM7) receptor CD97 is required for neutrophil [33] Jones, J., Krag, S.S. and Betenbaugh, M.J. (2005) Controlling N-linked glycan migration and host defense. J. Immunol. 172, 1125–1131. site occupancy. Biochim. Biophys. Acta 1726, 121–137.

40 N-glycosylation regulates the GPS auto-proteolysis

Supplementary data

Fig. S1. Analysis of GPS proteolysis of the wild type and mutant CD97 receptor molecules. Western blot analysis of total cell lysate from cells expressing the wildtype CD97 receptor (lane 2) and mutant receptors, CD97-G2,5 (lane 3), CD97- G1,4,5 (lane 4), CD97-G1-5 (lane 5), and CD97-S531A (lane 6). Lane 1 is mock control. The blot was probed with anti-myc fpr CD97 cleavage analysis and antibeta- actin for loading control. Note that the wild-type CD97, CD97-G2,5, and CD97- G1,4,5 all showed the uncleaved (~76-100 kDa) and cleaved fragments (~25 kDa).A The CD97-G1-5 and CD97-S531A mutants were not cleaved. B All uncleaved Nglycosylation mutants migrated faster that the uncleaved wild-type receptor, indicating functional N-glycosylation at the mutated sites. The CD97-S531A mutant remains glycosylated, therefore appeared fuzzy and bigger in size.

A B

Fig. S2. Detection of GPS auto-proteolytic activities in CD97. (A) Different amounts (1, 3, and 6 g) of affinity- purified CD97-mFc proteins were digested without or with PNGase and neuraminidase and visualized by Coomassie brilliant blue staining. (B) CD97-mFc molecules with or without the PNGase and neuraminidase treatment were subjected to Western blot analysis probed by anti-mFc mAb.

41 A A

B B

Fig. S3. Quantitative analysis of the GPS autoproteolytic activities of various adhesion-GPCR mFc-fusion proteins. (A) The relative intensity of the uncleaved and cleaved forms of the EMR2-mFc, CD97-mFc and domain-swapping fusion proteins as detected by Western blotting was determined by densitometer. The sum of intensities of both cleaved and uncleaved bands was set as 100%. (B) The relative intensity of the uncleaved and cleaved forms of the EMR2-mFc, EMR3-mFc, EMR4-mFc and CD97-mFc fusion proteins.

A A

B B

Fig. S4. Quantitative analysis of the GPS autoproteolytic activities of various CD97-mFc proteins including the WT and Nglycosylation site mutants. (A) The relative intensity of the uncleaved and cleaved forms of CD97-mFc WT- and single Nglycosylation site mutant proteins. (B) The relative intensity of the uncleaved and cleaved forms of CD97-mFc multiple Nglycosylation site mutant proteins.