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The Journal of Immunology, 2001, 166: 1433-1438.
Copyright © 2001 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Granzyme B Proteolysis of a Neuronal Glutamate Receptor Generates an Autoantigen and Is Modulated by Glycosylation1

Lorise C. Gahring2, Noel G. Carlson, Erin L. Meyer and Scott W. Rogers2

Geriatric Research Education and Clinical Center, Salt Lake City Veterans Affairs Medical Center and University of Utah School of Medicine, Salt Lake City, Utah, 84112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Autoimmune processes are initiated when tolerance to self-proteins fails to be established or maintained and immune cells are stimulated by self-Ags. Although intracellular autoantigens are common, the origin of extracellular autoantigens is poorly defined and possibly more dangerous. In this study, we considered a mechanism for the origin of an extracellular autoantigen from the neuronal glutamate receptor subunit 3 (GluR3) in Rasmussen’s encephalitis, a severe form of pediatric epilepsy. We demonstrate that specific cleavage of GluR3 by granzyme B (GB), a serine protease released by activated immune cells, can generate the GluR3B autoantigenic peptide, but only if an internal N-linked glycosylation sequon within the GluR3-GB recognition sequence (ISND*S) is not glycosylated. However, this N-glycon sequon while glycosylated normally is inefficiently used and glycosylation can fail. These results suggest that GB/N-glycon sites may escape normal tolerance mechanisms and contribute to autoantibody-mediated immune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The generation of immune responsiveness to self-Ags can result in pathogenic autoimmune damage of tissues mediated by both humoral and cellular immune processes. Disease states ranging from rheumatoid arthritis to multiple sclerosis indicate that the process of autoimmunity can involve many types of self-Ags and can affect the function of all organs/tissues of the body. One aspect of autoimmunity that has proven difficult to study has been the identification of the self-Ags to which immune responses are generated and rationale as to how these particular Ags are targeted. Recent evidence has emerged (1, 2, 3) for the generation of novel intracellular autoantigens during apoptosis. Activated T lymphocytes and NK cells release proteolytic enzymes into target cells, which subsequently induce caspase activation and cell death. Granzyme B (GB),3 a serine protease (4), is one of the enzymes released into the target cell that induces caspase activation. Rosen and Casciola-Rosen (3) have demonstrated that serum from patients with autoimmune diseases harbors autoantibodies that react with proteolytic fragments resulting from GB cleavage of intracellular proteins. However, GB is also present extracellularly during inflammatory episodes as demonstrated by detection of this protease in the plasma and synovial fluid of individuals with inflammatory disease (5, 6). Implications for the presence of proteolytic enzymes in the extracellular milieu with regard to the generation of extracellular autoantigens was investigated in this report using an autoantigen of the CNS.

Abs to the neuronal glutamate receptor subunit 3 (GluR3) are found in the serum of children with the rare and severe form of pediatric epilepsy, Rasmussen’s encephalitis (RE) (7, 8, 9). RE is characterized in the brain by the presence of microglial nodules and perivascular lymphocytic infiltrates (10). We have found (7, 9, 11) that certain RE autoantibodies have the novel property of activating GluRs, and their removal correlates with clinical improvement (7). Analysis of GluR3 through deletion mapping and alanine-substitution mutagenesis defined the antigenic region targeted by agonist-like anti-GluR3 Abs as aa 372–388 (termed GluR3B) and also identified key residues in the receptor-activating epitope that are aligned on the surface of the folded peptide (7, 11, 12). Immunization of rabbits (7) or mice (13, 14) with GluR3B is sufficient to generate RE-like pathology and GluR3-activating Abs with properties equivalent to agonist-like RE autoantibodies (7, 11, 13, 14). Hence, GluR3B contains the amino acids essential for the production of GluR-activating Abs in animal models of RE and autoantibodies found in patients with RE.

What makes GluR3B a target of autoantibodies? Notably, the last four amino acids of the GluR3B autoantigen, residues 385–388 (ISND), comprise a potential GB cleavage sight (15) that is not conserved in other closely related GluR subunits (11, 16). In this report, we show that the GluR3B autoantigen is generated by GB cleavage but only in the absence of glycosylation at an N-glycosylation sequon internal to the GB recognition sequence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
GB proteolysis

GB (sp. act., 23.4 U/µg; Alexis Biochemicals) in GB buffer (10 mM HEPES, 2 mM EDTA, 1% Nonidet P40, and 5 mM DTT, pH7.4) was used in all experiments. HEK293 cells or cultured murine cortical neuronal cells (17) were solubilized with GB buffer. Crude membranes from mouse hippocampi (C57BL/6 mice) were prepared as described previously (18) before homogenization in GB buffer. GB buffer (without DTT) was used for deglycosylation with N-glycosidase F (NGlyF; 10 U/ml, 25,000 U/mg; Sigma, St. Louis, MO) or O-glycosidase (10 U/ml, 2.5 U/mg; Boehringer Mannheim, Indianapolis, IN). To inactivate any endogenous caspase activity, 5 mM iodoacetamide was added to the GB buffer immediately before sample isolation. DTT (5 mM) was added to lysates before GB if not already present in the lysis buffer.

Western blot detection of GluR3 and mutations of GluR3

The mAb mAB-2F5 is specific to the GluR3B peptide (ANEYERFVPFSDQQISNDAAC; alanines were added as spacers and one cysteine for coupling to BSA for immunogen in mice) and will recognize only the portion of the GluR3 protein containing this sequence following GB proteolysis. The specificity of this IgG2a mAb has been revealed by alanine-substituted GluR3B peptide-ELISA (11) to interact principally with residues Y374, R376, V378, P379, and F380 that are aligned on the folded peptide surface (12). For detection of GluR3B-containing proteins by Western blot analysis, blots were blocked in PBS with 5% dried milk and 0.05% Tween 20 before incubating overnight in mAB2F5. Peroxidase-labeled anti-mouse secondary Ab (Jackson ImmunoResearch, West Grove, PA) and ECL (Amersham, Arlington Heights, IL) were used for detection. Mutations were introduced into GluR3(flip) as described previously (11). Clones containing mutations were confirmed by automated DNA sequencing.

Identification of GB sites in GluR3 and other membrane proteins

The recognition sequence for cleavage by GB has been described in detail by Harris et al. (15). From these data, we derived a GB consensus sequence (P4 = I or V, P3 = no R, K, or P, P2 = no R, or K, and P1 requires D). The Oxford Molecular Omiga software Prosite program was used to search human receptor protein sequences for these sites (sequences using brackets indicate residues that are allowed at the designated location and braces indicate disallowed residues). The algorithms were for a strict GB site, [I,V]-{R,K,P}-{R,K}-[D] and for a less strict definition, [I,V,L,T,A,F]-{R,K,P}-{R,K}-[D]. To identify GB sequences with an internal N-glycosylation sequon, the formula was modified accordingly (e.g., for a strict GB site; [I,V]-{R,K,P}-[N]-[D]-[S,T]). Sequences searched were human ligand-activated ion channels and a subset of metabotropic receptors (i.e, muscarinic and 5-hydroxytryptamine (serotonin)) from the GenBank-National Center for Biotechnology Information database (140 unique human receptor protein sequences totaling 107,979 aa). For GB sites containing an internal N-glycosylation consensus sequence, the sequence was located by visual inspection as intracellular or extracellular according to currently available transmembrane models. To assign a probability of finding these sequences by chance, the expected probability of a GB site was estimated using the values for amino acid occurrence in proteins (19) as follows in percent: A, 8.3; R, 5.7; N, 4.4; D, 5.3; I, 5.2; L, 9.0; K, 5.7; F, 3.9; P, 5.1; S, 6.9; T, 5.8; Y, 3.2; and V, 6.6.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GB proteolysis of GluR3 fusion protein and GluR3 expressed by neurons

We first determined whether a portion of GluR3 containing the GluR3B region was a substrate for GB. A 212-aa fragment of GluR3 containing the GluR3B sequence was produced as a soluble trpE-GluR3 bacterial fusion protein (7) and coincubated with GB for 1 h at 37°C. GluR3 protein was visualized by Western blot analysis using the GluR3B-specific mAB-2F5 (see Materials and Methods). A fragment containing GluR3B of the size expected for GB cleavage of this fusion protein at the GluR3B site was generated (Fig. 1GoB) that was both GB concentration (Fig. 1GoB) and incubation time dependent (Fig. 1GoC). All proteolysis was inhibited in this, and remaining experiments, by the GB competitive inhibitor Ac-IETD (Fig. 1GoD).



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  		FIGURE 1. GluR3 contains a GB cleavage site at the autoreactive epitope associated with autoimmune RE as determined by Western blot analysis. A, Shown is a diagram of GluR3 that locates the GluR3B autoantigen (gray box; aa 372–388), adjacent sequence (gray) not required as immunogen or for Ab binding, and F380 (asterisk) to which binding by Ab correlates with agonist efficacy and subunit specificity (11 ). The dark underline identifies the GB recognition sequence and cleavage site (labeled arrow) and the N387 (outlined) that is in the context of a consensus N-glycosylation sequon (NDS). Another N-glycosylation sequon (NRT) is also noted. Notably, the mouse GluR3B differs from the human GluR3 sequence at one residue where serine 390 is an alanine. Proposed transmembrane domains (I, II, and III), re-entry loop (RL), and leader sequence (LS) are indicated. Other consensus N-glycosylation sequons are also noted (Y). B, An extracellular portion of rat GluR3 (aa 245–457) expressed as a soluble path-trpE-GluR3 fusion protein of 52,000 kDa was incubated with GB (0.5–20 nM) for 1 h at 37°C. GB cleavage at GluR3B removes ~7 kDa of fusion protein to generate the predicted 45-kDa fragment (arrow in B–D). Quantitation by densitometry of the product is shown. C, Time-dependent proteolytic cleavage of trpE-GluR3 fusion protein incubated for 1–60 min with 20 nM GB is shown and quantitated as described for B. D, Preincubation of GB with 100 nM Ac-IETD-CHO (Alexis Biochemicals) inhibits GB-mediated trpE-GluR3 cleavage. E, GB cleavage of GluR3 expressed by cultured neurons was incubated (1 h, 37°C) with or without added GB (100 nM) and/or NGlyF (1 h, 37°C). The decrease in GluR3 molecular mass reflects N-glycan removal and the arrowhead indicates the expected 43-kDa GB cleavage product. F, Deglycosylation of mouse brain GluR3 by NGlyF, but not O-glycosidase (Ogly), renders it a GB substrate. C57BL/6 mouse hippocampal crude membranes were treated as indicated. The arrowhead points to multiple GB cleavage products of GluR3 that are ~43 kDa. The additional bands varied between experiments and various lots of NGlyF. The open arrowhead points to the prominent 79,000-kDa band that is a GluR3 endogenous proteolytic fragment that occurs in all full-length GluR3 protein preparations, including transfected HEK293 cells.

 
Cell lysates from primary cortical neuronal cultures were incubated with 100 nM GB for 1 h. No cleavage was observed unless lysates were first incubated with NGlyF (1 h, 37°C) to remove N-glycans (Fig. 1GoE). Similar results were obtained with hippocampal lysates (18) prepared from C57BL/6 mice (Fig. 1GoF). Preincubation with O-glycosidase to remove O-glycans did not alter GB cleavage of GluR3 (Fig. 1GoF). Notably, preincubation with endoglycosidase H did not render GluR3 susceptible to GB cleavage (data not shown), suggesting that removal of the entire glycosylation tree is required for GB proteolysis. Addition of iodoacetamide, an inhibitor of caspases (1), did not inhibit GB cleavage of neuronal GluR3 (cultured or hippocampal), suggesting that activation of other caspases by GB is not involved. Further, incubation of GluR3 with purified caspase-3 does not generate cleavage products (data not shown).

Site-directed mutagenesis of GluR3 and expression in transfected cells

To confirm the identity of the amino acids in GluR3B required for GB cleavage and inhibition by N-glycans, we performed site-directed mutagenesis (11) followed by expression of these constructs in HEK293 cells. Wild-type GluR3 was transiently transfected into HEK293 cells (7) that were harvested 24–48 h later. No GluR3 immunoreactivity was observed in untransfected cells or cells transfected with green fluorescent protein (data not shown). As shown in Fig. 2Go, transfection of GluR3 produced a glycosylated GluR3 protein whose molecular mass decreased upon removal of N-glycans with NGlyF. GB incubation with deglycosylated GluR3 generated a proteolytic fragment consistent in molecular mass with the GluR3B fragment from neurons. Because GB sites require an aspartic acid (D) at P1, GluR3B residue D388 was mutated to an alanine (D388A). Wild-type or GluR3(D388A) was expressed transiently in HEK293 cells for 24–48 h, harvested in GB lysis buffer, and the samples were divided into equal portions for incubation with either NGlyF and then 100 nM GB, or with GB alone. GluR3(D388A) was no longer a substrate for cleavage by GB even upon deglycosylation (Fig. 2Go). The importance of N-glycans to protection from GB cleavage was demonstrated through substitution of GluR3(N387) with a valine GluR3(N387V) to abolish the GB internal N-glycan sequon. When this mutant GluR3 was expressed in HEK293 cells, it was susceptible to GB cleavage without preincubation with NGlyF (Fig. 2Go). To test whether the location of the N-glycosylation sequon is important for inhibition of GB cleavage, the double mutant GluR3(N387V, S389N) was constructed which moves the N-glycosylation sequon to the carboxyl side of GluR3(D388). As shown in Fig. 2Go, GluR3 (N387V, S389N) was cleaved by GB without prior deglycosylation. The results demonstrate that introducing a glycosylation site immediately adjacent to the P1 aspartic acid, but outside of the GB site, has no effect on GB cleavage.



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  		FIGURE 2. Western blot analysis demonstrates that GB cleavage of GluR3 at GluR3B is blocked by internal N-glycans. Wild-type GluR3 or mutated GluR3 was expressed transiently in HEK293 cells. The arrowhead (left panel) points to the GB cleavage product of transfected wild-type GluR3 whose mobility is identical to neuronal GluR3. GluR3(D388A) was not a substrate for GB cleavage (middle panel). GluR3(N387V), which removes the N-glycosylation sequon internal to the GB cleavage site, renders this form of GluR3 susceptible to cleavage by GB without prior NGlyF treatment (right panel, arrow; left two lanes). Notably, the larger GB cleavage product of GluR3(N387V) is reduced to the 43-kDa fragment by NGlyF (data not shown). Reintroduction of an N-glycosylation sequon to the carboxyl side of D388 (outside of the GB site) to create the double mutant GluR3(N387V, S389N) did not alter the ability of GB to directly cleave this substrate (right two lanes). The GB recognition sequences are underlined and N-glycosylation sequons are indicated by gray overhead bars.

 
Inefficient glycosylation and susceptibility to GB proteolysis

Not all consensus N-glycon sequons are used equally during cotranslational modification. Kesturi and colleagues (20, 21, 22) report that an aspartic acid between the asparagine and serine of the N-glycosylation sequon strongly reduces glycosylation efficiency. With the exception of tryptophan and proline, all other residues in this central position have little effect on glycosylation. However, this site is efficiently glycosylated if a threonine replaces the serine in the third position (20, 21, 22). Therefore, because the GluR3(D388) is required for GB recognition, and this amino acid resides between the asparagine and serine of this GluR3B glycosylation sequon, this could render GluR3 incompletely glycosylated at this site. We determined whether failure to glycosylate reveals a GB site and whether conversion of this site to a preferred N-glycosylation sequon alters GluR3 susceptibility to GB cleavage. Although GB cleavage of wild-type GluR3 is not detected following relatively short incubation times (1–2 h) without first treating with NGlyF (Fig. 1Go), if reactions are allowed to proceed for longer periods (4–24 h at 37°C), some iodoacetamide-insensitive (caspase-independent) cleavage is detected (Fig. 3GoA). This is consistent with the expectation that not all GluR3 is glycosylated at residue N387. However, if this site is changed to the threonine-containing N-glycosylation sequon, GluR3(S389T), no cleavage product is detected even following prolonged incubation with GB (Fig. 3GoA). Preincubation of GluR3(S389T) with NGlyF and then GB results in cleavage similar to that of wild type (data not shown). This result is consistent with the conclusion that the N-glycosylation sequon within the GB cleavage sequence of GluR3B may not always be glycosylated. If true, then some nonglycosylated GluR3 might also be expressed by neurons. To test this, neuronal membranes were subjected to prolonged incubation with GB without prior deglycosylation. As shown in Fig. 3GoB, there is an accumulation of GB cleavage product after at least 4 h of incubation, consistent with the product obtained from direct cleavage by GB of GluR3(N387V) (Fig. 3GoA). No degradation is observed in the absence of added GB (Fig. 3GoB). These results demonstrate that, although GluR3 contains a GB site that is protected by N-glycosylation, low levels of nonglycosylated GluR3 at the GluR3B-GB site exist. Consequently, a relatively small subfraction of GluR3 expressed in the brain appears to escape N-glycan addition at the GluR3B site, and the efficiency of this cotranslational modification determines the susceptibility of GluR3 to GB cleavage.



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  		FIGURE 3. N-glycosylation efficiency determines susceptibility of GluR3 to GB cleavage as determined by Western blot analysis. A, HEK293 cells expressing mutant GluR3(N387V) (VDS), wild-type GluR3 (NDS) or GluR3(S389T) (NDT), respectively, were incubated with GB for 4 h without prior deglycosylation. GB cleaves GluR3N387V efficiently (arrowhead); however, under conditions of prolonged GB incubation, a small amount of GB cleavage of wild-type GluR3 is detected. No GB cleavage of GluR3(S389T) is detected even when the incubation was extended for 24 h. B, Cultured cortical cells similarly incubated for prolonged times without (left) or with 100 nM GB (right) at 37°C. Arrowhead indicates the GB cleavage product expected if GluR3 is not glycosylated at the GluR3B N-glycosylation sequon. Black bars indicate the P1 (D) and P2 residues of the GB consensus sequence and gray lines indicate N-glycosylation sequons (NDS or NDT). Mutated amino acids are distinguished by outlining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The GluR3B autoantigen contains a GB site that overlaps with a relatively inefficiently used N-glycosylation sequon. Modifications in glycosylation have been noted to occur during inflammation (23, 24, 25); however, in the case of GluR3B, absence of the entire glycosylation tree appears to be required for GB cleavage, suggesting that inefficiency of glycosylation at this site is responsible for GB recognition. Both inflammation (especially viral infection) and trauma have long been viewed as candidates for initiating RE pathology, in part due to the close association with the onset of encephalitis, microglial nodules consisting of activated T cell infiltrates (26), and the subsequent appearance of clinical symptoms. The seemingly improbable convergence of possible disease-initiating factors may explain why RE and similar neuroautoimmune diseases are relatively rare (see Refs. 8, 27 for additional discussion).

Although circumstances leading to the lack of tolerance and generation of autoimmunity are unknown, cell death that results from DNA damage, growth factor deprivation, heat shock, oxidative stress, proimmune intracellular infection by bacteria or viruses, and inflammation is often closely associated. For example, during cellular apoptosis, caspase proteolysis of intracellular proteins appears to reveal protein fragments to which immune tolerance in the thymus is normally generated (2, 3). In this model, apoptotic cascades can be initiated following activation of NK cells and cytolytic cells and the release of GB that enters the target cell to activate caspases. However, GB itself can cleave susceptible intracellular proteins to generate novel fragments to which tolerance has not been generated and create potential targets of autoreactivity (2, 3). The relevance of GB sites in extracellular proteins to the generation of autoimmunity has not been explored. A search of 140 unique human surface receptor proteins reveals candidate GB sites to be quite common, occurring approximately every 170 aa (Table IGo). As such, it might be expected that many extracellular proteins would be targets of autoantibodies. However, known extracellular autoantigens are relatively infrequent, which may reflect a different mechanism than that proposed for the generation of intracellular autoantigens or that immune tolerance to these extracellular fragments already exists. In the present study, we suggest that the occurrence of a GB site coincident with an overlapping N-glycosylation sequon in the extracellular domain would be a strong autoantigen candidate. Notably, only 2 proteins in Table IGo contain such a strong site (including GluR3) and 10 other proteins contain sites considered as weak (Table IGo). Three of these proteins, the neuronal nicotinic acetylcholine receptor {alpha}7 subunit (nAChR{alpha}7), the voltage gated-calcium channel, and the voltage-gated potassium channel are members of receptor families known to be autoimmune targets in myasthenia gravis, Lambert-Eaton syndrome, and Isaac’s syndrome, respectively. The glycosylated GB sequence in the nAChR{alpha}7 subunit (VANDS) is located near the N terminus at residue 44, well away from the epitope commonly described as the main immunogenic region of the muscle nicotinic acetylcholine receptor which is the target of autoantibodies in most patients with myasthenia gravis. However, recent reports (28, 29) have described patients with autoantibodies toward both muscle and ganglionic nicotinic receptors that do not interact at the main immunogenic region. Since nAChR{alpha}7 is expressed by ganglionic neurons (30, 31) and contains strong sequence identity in this region with other neuronal and muscle nicotinic receptor subunits (data not shown), this region of the molecule would be a candidate for the location of the autoreactive epitope in these rarer cases. If true, as for RE, the susceptibility of this and other surface proteins to N-glycan-modulated GB cleavage may have important implications for both the generation and prediction of extracellular autoantigens.


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  		Table I. Identification of putative GB sites containing internal N-glycosylation (glycan) sequons in the extracellular protein domains

 


   Footnotes
 
1 This work was supported by National Institutes of Health Grant NS35181. Funding was also provided by Veterans Administration Merit grants (to L.C.G. and S.W.R), the Pew Charitable Trust (to S.W.R.), and the Browning Foundation of Utah. Back

2 Address correspondence and reprint requests to either Dr. Lorise C. Gahring or Dr. Scott W. Rogers, University of Utah School of Medicine, 15 North 2030 East, Room 2100, Salt Lake City, UT 84112-5330. Back

3 Abbreviations used in this paper: GB, granzyme B; GluR3, glutamate receptor subunit 3; RE, Rasmussen’s encephalitis; NGlyF, N-glycosidase F; nACHR{alpha}7, nicotinic acetylcholine receptor {alpha}7. Back

Received for publication November 2, 2000. Accepted for publication November 30, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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