Open Access

Giantin is the major Golgi autoantigen in human anti-Golgi complex sera

  • Kazuhisa Nozawa1,
  • Marvin J Fritzler2,
  • Carlos A von Mühlen3 and
  • Edward KL Chan1Email author
Arthritis Res Ther20036:R95

DOI: 10.1186/ar1035

Received: 29 September 2003

Accepted: 27 November 2003

Published: 15 December 2003


Anti-Golgi complex antibodies (AGAs) are primarily associated with systemic lupus erythematosus and Sjögren's syndrome. Here we report on the immunoreactivity of AGAs against five Golgi autoantigens (giantin, golgin-245, golgin-160, golgin-95/GM130, and golgin-97) and provide data from epitope mapping on the most common Golgi autoantigen, namely giantin. A total of 80 human sera containing AGAs, as defined by indirect immunofluorescence on HEp-2 cells, were analyzed by ELISA using recombinant autoantigens and immunoprecipitation. The proportion of AGA sera that reacted with the five Golgi autoantigens was correlated with the molecular mass of the Golgi antigens. Autoantibodies to giantin, the largest Golgi autoantigen, were the predominant AGAs, being found in 50% of the AGA sera. Epitope mapping of giantin was performed using six recombinant fragments spanning the entire protein. Antigiantin-positive sera with low titer autoantibodies recognized epitopes in the carboxyl-terminal fragments that are proximal to the Golgi membrane, whereas higher titer sera exhibited strong reactivity to amino-terminal and central domains that are likely to extend from the Golgi membrane into the cytoplasm. Our working hypothesis is that aberrantly expressed Golgi complex autoantigens may be released into the immune system when cells undergo lysis. By virtue of a carboxyl-terminal transmembrane domain, giantin is likely to be more stably associated with the cytoplasmic face of the Golgi complex than are other golgins, which are peripheral proteins. The stable association of giantin with the putative released Golgi complex may contribute to its preferential autoantigenicity.


anti-Golgi complex antibody autoantibody autoimmunity cell death epitope mapping


The Golgi complex is an elaborate cytoplasmic organelle that has a prominent function in the processing, transporting, and sorting of intracellular proteins subsequent to their synthesis in the rough endoplasmic reticulum. Structurally, the Golgi complex is localized in the perinuclear region of most mammalian cells and is characterized by stacks of membrane-bound cisternae, as well as by functionally distinct trans-Golgi and cis-Golgi networks [1]. Interestingly, several Golgi proteins have been reported to be targets of the autoimmune response, even though they are localized to the cytoplasmic face of Golgi membranes, a site that is presumed to be privileged in that it is protected from immune surveillance. Autoantibodies directed against the Golgi complex were first identified in the serum of a Sjögren's syndrome patient with lymphoma [2]. Several isolated reports have described anti-Golgi complex antibodies (AGAs) in other systemic autoimmune diseases such as systemic lupus erythematosus (SLE) [3], rheumatoid arthritis [4], mixed connective tissue disease [5], and Wegener's granulomatosis [6]. AGAs were also found in 10% of patients with HIV infection [7] and 35.7% of HIV carriers [8]; however, in the more recent report by Massabki and coworkers [9], AGAs were not found in 100 HIV-infected patients.

Within the past several years, our laboratories and others have cloned and identified several novel Golgi autoantigens. This has been achieved primarily by expression cloning using human autoantibody probes. These Golgi autoantigens are referred to as giantin/macrogolgin/GCP372, golgin-245/p230, golgin-160/GCP170, golgin-95/GM130, golgin-97, and golgin-67, with their names based in part on their molecular weights as estimated from SDS-PAGE under denaturing conditions [7, 1013]. A common feature of this family of Golgi autoantigens is that they all have coiled-coil domains throughout the entire protein except for short nonhelical regions at the amino-terminus and carboxyl-terminus [1]. Golgin-245 was localized to the trans-Golgi compartment [14], whereas GM130 has been reported to be localized to the the cis-Golgi compartment [15]. It has been also reported that several golgins, such as golgin-245 and golgin-97, are attached to Golgi membranes through a GRIP domain in the carboxyl-termini [16]. In contrast to other Golgi autoantigens, giantin has a single transmembrane domain in the carboxyl-terminus [17]. A second common feature among the Golgi autoantigens is that biochemical evidence and immunoelectron microscopy data show that they are peripheral or transmembrane (giantin) proteins on the cytoplasmic face of the Golgi complex. The implication is that these Golgi autoantigens may have common biochemical characteristics and functions that make them preferred autoimmune targets among the approximately 100 Golgi complex proteins described to date [18]. A third common feature among the Golgi autoantigens is that none of these macromolecules are localized to apoptotic blebs [19]; in fact, immunofluorescence analysis showed that the Golgi complex was altered and developed distinctive characteristics during apoptosis and necrosis [19].

It is striking that human autoimmune responses are selective for these proteins that are rich in coiled-coil motifs and that reside on the cytoplasmic face of the Golgi complex. How this family of coiled-coil proteins becomes autoimmune targets remains to be determined. One possible explanation is that these Golgi proteins may be recognized as surface structures on the organelle that is exposed to the immune system in aberrant disease states associated with unregulated cell death (apoptosis and necrosis) resulting from injury or infection, and defective clearance of dying cells.

Although it is known that AGAs are associated with some autoimmune diseases or viral infections, the prevalence of AGAs and their fine specificity have not been reported. Immunoblotting and immunoprecipitation studies have shown that AGAs reacted with a number of cellular proteins [20]. AGAs are generally considered to be rare autoantibodies; however, Bizzaro and coworkers [21] suggested that the presence of AGAs in high titer in the absence of a clear clinical manifestation may constitute an early sign of systemic autoimmune diseases. Here, we present data on the reactivity of AGAs against known Golgi autoantigens by ELISA using five recombinant proteins. Because antigiantin autoantibodies were found to be the most common reactivity in AGAs, epitope mapping was performed using six overlapping recombinant fragments of giantin.

Materials and method

Human sera and monitoring of anti-Golgi complex antibody reactivity

Human putative AGA sera and normal control sera were obtained from the laboratory serum bank and Advanced Diagnostics Laboratory at the University of Calgary, Canada. Some AGA sera were also provided by Drs R L Humbel (Luxembourg), Kiyomitsu Miyachi (Keigu Clinic, Yokohama, Japan), and Carlos A von Mühlen (Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil). All sera were provided as anonymous samples and were stored at -80°C until use. The reactivity to Golgi complex in all AGA sera was confirmed by indirect immunofluorescence (IIF) microscopy on HEp-2 cells (Immuno Concepts Inc., Sacramento, CA, USA). Double staining was performed using the human sera (1 : 100 dilution) and rabbit antigiantin antiserum (1 : 500 dilution) as a marker of the Golgi complex [19]. The secondary antibodies were Alexa Fluor® 488 conjugated goat antihuman IgG reagents and Alexa Fluor® 568 conjugated goat antirabbit IgG reagents (Molecular Probes, Eugene, OR, USA) used at a dilution of 1 : 400. Nuclei were counterstained with 4',6-diamidino-2-phenylindole. By using this approach, a total of 80 sera exhibited specific staining of the Golgi complex.

Recombinant Golgi proteins

Recombinant human Golgi autoantigens were produced using the expression plasmid pET28 system in Escherichia coli BL21 (DE3; Novagen, Madison, WI, USA) as previously described [19]. Recombinant proteins of golgin-245 (amino acids 811–2083) [11], golgin-160 (amino acids 787–1348) [10], golgin-95/GM130 (amino acids 370–990) [10], and golgin-97 (amino acids 1–767) [12] were subcloned into pET28 vectors for the expression of recombinant bacterial proteins. Six overlapping fragments P1–P6 representing the full-length giantin cDNA (GenBank accession number NM_004487) [7] were generated for epitope mapping analysis. Two fragments (P1 and P2) were obtained by expression cloning from a random-primed lambda phage cDNA library generated from human T24 cells using an antigiantin-specific human serum. Three fragments were obtained from an available expression sequence tag clone (P3, GenBank accession number N_76853; P4, GenBank accession number BG_567238; P5, GenBank accession number AI_458639). One fragment (P6) was cloned from reverse transcription polymerase chain reaction synthesis using total RNA purified from HeLa cells. All six fragments of overlapping recombinant proteins cDNAs were inserted into pET28 expression vector and introduced into Escherichia coli BL21 (DE3). Sequencing was conducted in both directions using custom primers. Bacterial pellets were suspended in 6 M guanidinium hydrochloride containing buffer, and the recombinant proteins were purified by nickel column chromatography according to manufacturer's instructions (Qiagen, Valencia, CA, USA). The concentration of the purified recombinant proteins was measured by a Protein DC Assay Kit (Bio-Rad, Hercules, CA, USA) and these samples were stored at -80°C until they were required for subsequent experiments.

Enzyme-linked immunosorbent assay

The ELISA protocol described by Rubin [22] was used with some modifications. In brief, Ni column affinity purified recombinant proteins were diluted in phosphate-buffered saline to a final concentration of 1 μg/ml and then coated on Immulon 2 microtiter plates (Dynatech Laboratories, Alexandria, VA, USA). Human sera were diluted 1 : 1000 and then incubated in the antigen-coated wells. Horseradish peroxidase-conjugated goat antihuman IgG (CALTAG Laboratories, San Francisco, CA, USA) was used at 1 : 5000 dilution and the substrate 2,2'-azinobis (3-ethylbenzthiazoline) sulfonic acid was added as the detection reagent. Each sample was analyzed in duplicate and the average optical density (OD) at 405 nm with a substrate development time of 15–45 min was used for data analysis. The cutoff value designating a positive reaction was the mean OD of 12 normal sera +3 standard deviations (SDs).


HeLa cells (ATCC, Rockville, MD, USA) were metabolically labeled overnight with [35S]-methionine (Trans 35S-label; ICN), as described previously [11, 12]. Cell extracts were harvested in a lysis buffer containing 1% NP-40, 50 mmol/l Tris.HCl, pH 7.5 and 150 mmol/l NaCl, and supplemented with Complete™ protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN, USA). Soluble fractions were used as substrate for immunoprecipitation reactions by combining 100 μl 10% protein A-Sepharose beads (Sigma, St. Louis, MO, USA), 10 μl human serum, 500 μl NET2 buffer (50 mmol/l Tris-HCl, 150 mmol/l NaCl, 5 mmol/l EDTA, 0.5% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 0.02% sodium azide, pH 7.4), and 50–100 μl labeled cell extract. After 1 hour of incubation at 8°C, the Sepharose beads were washed five times in NET2. Proteins were eluted in 20 μl sample buffer and analyzed by 10% gel SDS-PAGE [23], followed by autoradiography.


Affinity purified recombinant proteins were loaded on 12.5% SDS-PAGE gels (4 μg/lane), separated by electrophoresis, and transferred to nitrocellulose membranes using a Semi-Dry Trans-Blot apparatus (Bio-Rad), as described previously [19]. Human sera containing antigiantin antibodies were used at dilutions of 1 : 100 to 1 : 500. Detection of bound antibodies was achieved using horseradish peroxidase-conjugated goat antihuman IgG antibody (CALTAG Laboratories), used at 1 : 5000 dilution, in combination with enhanced chemiluminescence (Super Signal; PIERCE Products, Rockford, IL, USA).


Giantin is the most common autoantigen detected in anti-Golgi complex antibody sera

Reactivity to Golgi complex antigens in all sera was confirmed by IIF, and all sera exhibited a specific staining pattern against Golgi complex structures as determined by colocalization with rabbit antibodies to giantin (Fig. 1). This approach yielded 80 human AGA sera that were used to investigate the prevalence of autoantibodies to five individual Golgi autoantigens represented by purified recombinant proteins in an ELISA and by immunoprecipitation using extracts from [35S]-methionine labeled HeLa cells (Fig. 2). The number of positive sera and frequency of reactivity of the 80 human AGA sera are summarized in Table 1. The most common Golgi complex autoantigen target was giantin (40/80 [50%]) and the second most common target was golgin-245 (19/80 [24%]). The lowest frequency reactivity (3.8%) was to golgin-97, and 25 AGA sera (31.3%) did not react with any of the five Golgi autoantigens used in the present study. Interestingly, the frequency of AGA sera reactive with the five Golgi autoantigens was numerically correlated with the molecular masses of the native Golgi autoantigens (Table 1), and the number of positive sera that reacted with giantin was remarkably higher than those for other golgins. The confirmation by immunoprecipitation was important because some of the Golgi autoantigens used as substrate in the ELISA did not represent full-length proteins and we were concerned that reactivity to these five Golgi autoantigens may be underestimated by ELISA alone. Among the 25 AGA sera that were negative for the five Golgi autoantigens, there were no predominant reactivities other than the five Golgi autoantigens described, even though the immunoprecipitation assay showed unidentified bands that were recognized by many of these sera.
Figure 1

Immunofluorescence colocalization of putative human anti-Golgi complex antibody (AGA) sera with rabbit antigiantin antibody to confirm specificity to the Golgi complex. (a) A representative human serum exhibiting staining specific to the Golgi complex in HEp-2 cells colocalizes with (b) characteristic staining by rabbit antigiantin antibody (arrows). (c) A serum with diffuse nuclear and cytoplasmic staining unrelated to the Golgi complex as demonstrated by (d) lack of costaining with rabbit antigiantin antibody.
Figure 2

Representative data from the immunoprecipitation analysis of anti-Golgi complex antibody (AGA) using extracts from HeLa cells metabolically labeled with [35S]-methionine for 16 hours. Lane 1, normal human serum; lanes 2–5, AGA sera. Lanes 2 and 3 show sera with primarily antibody to golgin-160 (g160) and giantin, respectively. Lane 4 shows a serum with antibodies to giantin and golgin-97 (g97). Lane 5 shows a serum with antibodies to giantin, golgin-245 (g245), gm130, and an unknown protein (arrowhead) migrated at approximately 90 kDa. Lane 6 shows a serum with strong reactivity to golgin-245 and weaker reactivity to several unidentified lower molecular weight proteins (*).

Table 1

Frequency of autoantibodies to specific Golgi complex autoantigens in 80 human anti-Golgi complex antibody sera

Golgi autoantigen (molecular weight [kDa])

Positive sera (n [%])

Giantin (370)

40 (50.0)

Golgin-245 (245)

19 (23.8)

Golgin-160 (160)

11 (13.6)

Golgin-95/GM130 (130)

6 (7.5)

Golgin-97 (97)

3 (3.8)

Undefined anti-Golgi reactivity

25 (31.3)

A total of 80 sera were studied.

Anti-Golgi complex antibody correlations

We then determined whether there were specific correlations between any of the five specific AGAs with another AGA. None of the sera had AGAs to four or more of these five Golgi autoantigens. There were 6, 15, and 34 sera with antibodies to three, two, and one of the five Golgi autoantigens, respectively. Among the six sera with antibodies to three of the five antigens, four sera had antigiantin, antigolgin-245 and antigolgin-160, which were the three most common antibodies detected; one serum had antigiantin, antigolgin-245 and antigolgin-95/GM130 (Fig. 2, lane 5); and the remaining serum had antigiantin, antigolgin-245, and antigolgin-97. Among the 15 sera with antibodies to two of the five antigens, five had antigiantin and antigolgin-245, two had antigiantin and antigolgin-160, two had antigiantin and anti-GM130, two had antigiantin and antigolgin-97, two had antigolgin-245 and antigolgin-160, and two had antigolgin-245 and anti-GM130 (Table 2). No specific correlations were observed between the two most abundant antibodies, namely antigiantin and antigolgin-245. For example, among the 19 AGA sera positive for antibody to golgin-245, 11 (57.9%) were positive and 8 (42.1%) were negative for antigiantin antibody. Among the 40 AGA sera positive for antibody to giantin, 11 (27.5%) were positive and 29 (72.5%) were negative for antigolgin-245. Although the number of sera that bound golgin-160, GM130, and golgin-97 were relatively small, it was interesting that sera with these three autoantibodies did not overlap. In other words, sera positive for antigolgin-160 were negative for antibody to GM130 and golgin-97, and sera positive for anti-GM130 were negative for antigolgin-97 (Table 2).
Table 2

Correlation of the five antibodies detected in the current study among the 80 anti-Golgi complex antibody sera analyzed


















Antigiantin (n = 40)


11 (27.5%)

29 (72.5%)

5 (12.5%)

35 (87.5%)

3 (7.5%)

37 (92.5%)

3 (7.5%)

37 (92.5%)

Antigolgin-245 (n = 19)

11 (57.9%)

8 (42.1%)


6 (31.5%)

13 (68.5%)

3 (15.9%)

16 (84.1%)

1 (5.2%)

18 (94.8%)

Antigolgin-160 (n = 11)

5 (45.5%)

6 (54.5%)

6 (54.5%)

5 (45.5%)



11 (100%)


11 (100%)

Anti-GM130 (n = 6)

3 (50%)

3 (50%)

3 (50%)

3 (50%)


6 (100%)



6 (100%)

Antigolgin-97 (n = 3)

3 (100%)


1 (33.3%)

2 (66.7%)


3 (100%)


3 (100%)


Characterization of major epitopes in giantin

To examine the relative distribution of epitopes in giantin, we performed mapping using six overlapping partial length constructs of recombinant giantin. Expression vectors for the recombinant proteins P1–P6 were constructed to cover the full-length of giantin (amino acids 1–3259), with the exception of the 38 amino acids at the amino-terminus (Fig. 3). Forty sera containing antigiantin antibody were analyzed using ELISA (Fig. 4, Table 3). Based on reactivity to the P1–P6 peptides, we provisionally divided the positive sera into low-positive and high-positive groups. Low-positive was defined as OD values between the mean OD of normal sera plus 3–15 SDs, whereas high-positive was defined as the OD greater than the mean OD of normal sera plus 15 SDs. Giantin is known to be bound on the cytoplasmic face of the Golgi complex via its single carboxyl-terminal transmembrane domain, and the amino-terminal and the central domains extend into the cytoplasm [24, 25].
Figure 3

Physical map of giantin cDNA fragments used in epitope analysis. The open box denotes the open reading frame. P1–P6 represent overlapping segments expressed as recombinant proteins, together spanning almost the full length of giantin. The P1 fragment differs from the published sequence and represents an alternatively mRNA spliced product. (b) Coiled-coil domains of giantin, golgin-245, golgin-160, golgin-95/GM130, and golgin-97. Each macromolecule is depicted showing the coiled-coil regions, as predicted by the COILS program [43]. A transmembrane (TM) hydrophobic region of 20–22 amino acids at the carboxyl-terminus is postulated to be responsible for anchoring giantin to Golgi membrane, with the molecule extending to the cytoplasm. The cytoplasmic domains of giantin are responsible for interaction with other Golgi proteins GM130 and p115.

Table 3

Epitope mapping of giantin

Recombinant fragments

Total positive sera (n [%])

Low-positive sera (%)

High-positive sera (%)

P1, aa 39–1040 (1001 aa)

12 (30.0)

7/12 (58.3)

5/12 (41.7)

P2, aa 851–1450 (599 aa)

11 (27.5)

8/11 (72.7)

3/11 (27.3)

P3, aa 1435–2204 (769 aa)

11 (27.5)

7/11 (63.6)

4/11 (36.4)

P4, aa 2019–2568 (549 aa)

7 (17.5)

3/7 (42.9)

4/7 (47.1)

P5, aa 2550–2843 (293 aa)

15 (37.5)

15/15 (100)


P6, aa 2818–3259 (441 aa)

22 (55.0)

22/22 (100)


A total of 40 antigiantin positive human sera were analyzed for reactivity in ELISA. Cutoff value for a positive reaction: the mean optical density (OD) of normal human sera +3 standard deviations (SDs). Low-positive: the OD between the mean of normal sera +3 SDs to +15 SDs. High-positive: OD greater than the mean of normal sera +15 SDs. aa, amino acids.
Figure 4

Reactivity of human antigiantin positive sera against six overlapping giantin fragments. A total of 40 antigiantin positive sera were analyzed by ELISA. Black squares represent optical density (OD) values for each individual serum. Dotted lines represent cutoff values for the low-positive group (between the mean OD of normal sera plus 3–15 standard deviations [SDs]), and dashed lines represent cutoff values for the high-positive group (greater than mean OD of normal sera +15 SDs).

None of antigiantin positive sera showed high positive reactivity to P5 or P6 peptides; however, the highest proportion of antibody reactivity (22/40 [55%]) was to P6, which includes the carboxyl-terminus and the transmembrane signal sequence. P5, which is proximal to the transmembrane domain and the cytoplasmic face of the Golgi membrane, also exhibited higher antibody frequency than those for other fragments more distal to the transmembrane domain (P1–P4). In contrast to anti-P5 and anti-P6, some AGA sera had antibodies to the distal fragments P1–P4 exhibiting high-positive reactivity, but the overall frequency of antibody to these fragments was relatively low (Fig. 4 and Table 3). All of the antigiantin sera reacted with one or more of the giantin subfragments used for epitope mapping. There were no specific correlations between P1–P4 high-positive sera (Fig. 4) and another coexisting AGA; among the P1–P4 high-positive sera, only one serum had coexisting antigolgin-160 and a second serum had coexisting anti-GM130. Taken together, these data suggest that the major epitopes of giantin are located in the carboxyl-terminal domain, including the transmembrane signal sequence. However, the epitopes localized in the distal amino-terminus or central domains of giantin can generate stronger autoimmune responses than can the epitopes in the transmembrane region.


In the present study we investigated the frequency of autoantibodies to specific Golgi complex autoantigens in a cohort of human sera containing AGAs as defined by IIF. The most frequent target autoantigen was giantin. Autoepitopes of giantin span across the entire protein, but the most frequent reactivity was located in the carboxyl-terminal fragments P5 and P6. These data are consistent with the earlier report by Seelig and coworkers [7] describing a diverse spectrum of AGAs that recognized different recombinant fragments in a smaller cohort of AGA sera. In contrast to antibodies to giantin, the least common AGAs were those directed at golgin-97, which also has the lowest molecular mass among the group of Golgi complex autoantigens included in the present study. The proportion of antibody to giantin was more than 10-fold greater than that to golgin-97.

To understand the mechanism of Golgi autoantibody production, it is important to consider why giantin has a greater frequency of reactivity than do other golgins. Differences between giantin and other golgins include the following: giantin is the highest molecular weight Golgi protein and contains a greater number of coiled-coil domain units than other golgins (Fig. 3b); and only giantin possesses a transmembrane domain, which may ensure its tight association with the Golgi complex.

Giantin is the most common target autoantigen in anti-Golgi complex antibody sera

Although we showed that the majority of sera that react with the Golgi complex in an IIF assay react with known Golgi autoantigens, 25 out of 80 (31.3%) AGA sera did not recognize any of the five Golgi autoantigens examined in this study. The data suggest that these sera react with other Golgi autoantigens. A candidate Golgi autoantigen is GMAP-210, a reported cis-Golgi network associated protein that also contains characteristic coiled-coil domains [26]. Among the 80 AGA sera, our immunoprecipitation data revealed three sera with a common band at approximately 210 kDa that might represent GMAP-210; however, because we did not have the cDNA for GMAP-210 and the frequency of this putative anti-GMAP-210 antibody was low, we did not confirm these data using independent methods. Two other Golgi proteins that may be candidate autoantigens include golgin-84, an 84 kDa transmembrane Golgi protein [27]; and βI Sigma spectrin, a 220 kDa protein that is associated with Golgi complex and vesicles [28]. However, our immunoprecipitation data did not yield any bands consistent with these Golgi complex candidate autoantigens. We did not include other known Golgi autoantigens such as golgin-67 [13] and p115 [29] because the frequencies of these autoantibodies are known to be low. Thus, our data support the notions that the five selected Golgi autoantigens are the most prevalent in AGA sera and that giantin is the most common Golgi autoantigen recognized in AGA sera.

Coiled-coil domain units may enhance selection as autoantibody targets?

The Golgi autoantigens identified to date are related because they have similar overall secondary structures, as evidenced by extensive coiled-coil rod domains in the central region and small non-coiled-coil or globular domains at both the carboxyl-terminus and amino-terminus [1]. The cumulative length of coiled-coil domains are thus directly proportional to the molecular mass of the Golgi autoantigens (Fig. 3b). For example, giantin clearly has more coiled-coil units than does golgin-97.

The human autoimmune response to Golgi autoantigens appears to be highly specific because many AGA sera react with only one (34/80 [42.5%]) or two (15/80 [18.8%]) of the five autoantigens. The specificity of the autoimmune response is demonstrated in the present study. For example, 23 of the 40 antigiantin positive sera reacted with giantin without coexisting autoantibodies to other five golgins. The lack of correlation with the frequency of antibody, as shown in Table 2, is consistent with the conclusion that it is unlikely that the immune response is merely directed at cross-reactive coiled-coils in these self-proteins. It is interesting to note that large (approximately 100 kDa or greater) coiled-coil rich proteins were noted in many non-Golgi cytoplasmic organelles, including endosomal protein EEA1 [30] and CLIP-170 [31], and the centrosomal proteins pericentrin [32], ninein [33], and Cep250 and Cep110 [34]. The mitotic organelles are also known to be associated with large coiled-coil rich autoantigens, including the mitotic apparatus proteins NuMA [35, 36] and centromere-associated protein CENP-E [37] and CENP-F [38]. It is noteworthy that we did not observe coexisting autoantibodies to these other coiled-coil rich organelles in our study of these AGA sera. These endosome, centrosome, and mitotic apparatus associated autoantigens are, like the golgins, proteins with high molecular masses and high content of coiled-coil domains. The combination of these two physical features in autoantigen may promote the induction and production of autoimmune antibody in certain disease states. As discussed above, this may have general significance in other autoantigens other than those associated with the Golgi complex.

Golgi autoantigens as surface structures on organelles released to the immune system

Another possible reason why giantin has a high frequency of reactivity among the Golgi autoantigens is that giantin is a somewhat unique Golgi complex autoantigen in that it possesses a transmembrane domain. It is not clear why and how the immune system is able to recognize or target these proteins because it is generally thought that the immune system is not exposed to intact intracellular self-antigens. One possible explanation is that they may be surface structures represented on cytoplasmic organelles that are recognized as foreign by the immune system in aberrant disease states associated with unregulated cell death (apoptosis or necrosis) resulting from injury or infection. A variety of autoantigens are cleaved into signature fragments during apoptosis and necrosis [39]. The emerging view is that the modified forms of autoantigens generated during cell death might stimulate autoantibody responses if presented to the immune system in a proinflammatory context [40]. We and others previously reported that distinct cleavage fragments of Golgi autoantigens were generated during apoptosis and necrosis, and we also observed that, compared with other golgins, giantin is readily cleaved into multiple fragments during apoptosis [19, 41]. Furthermore, we observed that the Golgi complex itself was also fragmented during apoptosis and necrosis [19]. It is interesting to note that, unlike 60 kDa SS-A/Ro and some other autoantigens targeted by autoantibodies from Sjogren's syndrome and SLE sera [42], golgins do not appear to be expressed on membranous apoptotic blebs [19]. One explanation for this apparent paradox may be the unique nature of the trans-membrane domain of giantin and the GRIP domain of other golgins that allow the presentation of these Golgi membrane-stabilized antigens to the immune system independently of apoptotic blebs. It is possible that giantin is more stably associated with the remaining Golgi surface membrane than other golgins by virtue of its transmembrane domain when cells undergo cell death. Because the cleaved Golgi autoantigens are antigenic [19, 41], they may play a role in sustaining autoantibody production in certain autoimmune disease states.


Our work and that of other investigators have shown that coiled-coil rich Golgi proteins are the predominant targets of human anti-Golgi autoantibodies. Here we showed that the most common Golgi autoantigen was giantin. Our data suggest at least two possible explanations for the production of human AGAs. One is that high molecular mass proteins with high content of coiled-coils induce heightened autoimmune responses. The other is that Golgi autoantigens may be recognized as surface structures on cytoplasmic organelles that are released to the immune system when cells undergo cell lysis. Giantin is likely to be more stably associated with remnants of Golgi fragments than other Golgi peripheral proteins, because only giantin has a transmembrane domain.



anti-Golgi complex antibody


enzyme-linked immunosorbent assay


indirect immunofluorescence


optical density


standard deviation


systemic lupus erythematosus.



This work was supported in part by National Institutes of Health Grants AI39645 and AI47859 (EKLC), and Canadian Institutes for Health Research Grant MOP-38034 (MJF).

Authors’ Affiliations

Department of Oral Biology, University of Florida College of Dentistry
Department of Biochemistry and Molecular Biology, University of Calgary
Department of Internal Medicine, Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul


  1. Chan EKL, Fritzler MJ: Golgins: coiled-coil proteins associated with the Golgi complex. Electronic J Biotechnol. 1998, 1: 1-10.View ArticleGoogle Scholar
  2. Rodriguez JL, Gelpi C, Thomson TM, Real FJ, Fernandez J: Anti-golgi complex autoantibodies in a patient with Sjögren syndrome and lymphoma. Clin Exp Immunol. 1982, 49: 579-586.Google Scholar
  3. Fritzler MJ, Etherington J, Sokoluk C, Kinsella TD, Valencia DW: Antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the Golgi apparatus. J Immunol. 1984, 132: 2904-2908.Google Scholar
  4. Hong HS, Morshed SA, Tanaka S, Fujiwara T, Ikehara Y, Nishioka M: Anti-Golgi antibody in rheumatoid arthritis patients recognizes a novel antigen of 79 kDa (doublet) by western blot. Scand J Immunol. 1992, 36: 785-792.View ArticleGoogle Scholar
  5. Rossie KM, Piesco NP, Charley MR, Oddis CV, Steen VD, Fratto J, Deng JS: A monoclonal antibody recognizing golgi apparatus produced using affinity purified material from a patient with connective tissue disease. Scand J Rheumatol. 1992, 21: 109-115.View ArticleGoogle Scholar
  6. Mayet WJ, Hermann E, Csernok E, Knuth A, Poralla T, Gross WL, Meyer zum Buschenfelde KH: A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener's granulomatosis. J Immunol Methods. 1991, 143: 57-68. 10.1016/0022-1759(91)90272-H.View ArticleGoogle Scholar
  7. Seelig HP, Schranz P, Schroter H, Wiemann C, Renz M: Macrogolgin: a new 376 kD Golgi complex outer membrane protein as target of antibodies in patients with rheumatic diseases and HIV infections. J Autoimmun. 1994, 7: 67-91. 10.1006/jaut.1994.1006.View ArticleGoogle Scholar
  8. Gentric A, Blaschek M, Julien C, Jouquan J, Pennec Y, Berthelot JM, Mottier D, Casburn-Budd R, Youinou P: Nonorgan-specific autoantibodies in individuals infected with type 1 human immunodeficiency virus. Clin Immunol Immunopathol. 1991, 59: 487-494. 10.1016/0090-1229(91)90043-A.View ArticleGoogle Scholar
  9. Massabki PS, Accetturi C, Nishie IA, da Silva NP, Sato EI, Andrade LE: Clinical implications of autoantibodies in HIV infection. AIDS. 1997, 11: 1845-1850. 10.1097/00002030-199715000-00009.View ArticleGoogle Scholar
  10. Fritzler MJ, Hamel JC, Ochs RL, Chan EKL: Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex. J Exp Med. 1993, 178: 49-62. 10.1084/jem.178.1.49.View ArticleGoogle Scholar
  11. Fritzler MJ, Lung CC, Hamel JC, Griffith K, Chan EKL: Molecular characterization of golgin-245: a novel Golgi complex protein containing a granin signature. J Biol Chem. 1995, 270: 31262-31268. 10.1074/jbc.270.52.31262.View ArticleGoogle Scholar
  12. Griffith KJ, Chan EKL, Lung CC, Hamel JC, Guo X, Miyachi K, Fritzler MJ: Molecular cloning of a novel 97-kd Golgi complex autoantigen associated with Sjögren's syndrome. Arthritis Rheum. 1997, 40: 1693-1702.View ArticleGoogle Scholar
  13. Eystathioy T, Jakymiw A, Fujita DJ, Fritzler MJ, Chan EKL: Human autoantibodies to a novel Golgi protein golgin-67: high similarity with golgin-95/gm 130 autoantigen. J Autoimmun. 2000, 14: 179-187. 10.1006/jaut.1999.0359.View ArticleGoogle Scholar
  14. Erlich R, Gleeson PA, Campbell P, Dietzsch E, Toh BH: Molecular characterization of trans-Golgi p230. A human peripheral membrane protein encoded by a gene on chromosome 6p12-22 contains extensive coiled-coil alpha-helical domains and a granin motif. J Biol Chem. 1996, 271: 8328-8337. 10.1074/jbc.271.14.8328.View ArticleGoogle Scholar
  15. Nakamura N, Rabouille C, Watson R, Nilsson T, Hui N, Slusarewicz P, Kreis TE, Warren G: Characterization of a cis-Golgi matrix protein, GM130. J Cell Biol. 1995, 131: 1715-1726. 10.1083/jcb.131.6.1715.View ArticleGoogle Scholar
  16. Munro S, Nichols BJ: The GRIP domain: a novel Golgi-targeting domain found in several coiled-coil proteins. Curr Biol. 1999, 9: 377-380. 10.1016/S0960-9822(99)80166-3.View ArticleGoogle Scholar
  17. Seelig HP, Schranz P, Schroter H, Wiemann C, Griffiths G, Renz M: Molecular genetic analyses of a 376-kilodalton Golgi complex membrane protein (giantin). Mol Cell Biol. 1994, 14: 2564-2576.View ArticleGoogle Scholar
  18. Taylor RS, Jones SM, Dahl RH, Nordeen MH, Howell KE: Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities. Mol Biol Cell. 1997, 8: 1911-1931.View ArticleGoogle Scholar
  19. Nozawa K, Casiano CA, Hamel JC, Molinaro C, Fritzler MJ, Chan EKL: Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis. Arthritis Res. 2002, 4: R3-10.1186/ar422.View ArticleGoogle Scholar
  20. Kooy J, Toh BH, Gleeson PA: Heterogeneity of human anti-Golgi auto-antibodies: reactivity with components from 35 to 260 kDa. Immunol Cell Biol. 1994, 72: 123-127.View ArticleGoogle Scholar
  21. Bizzaro N, Pasini P, Ghirardello A, Finco B: High anti-golgi autoantibody levels: an early sign of autoimmune disease?. Clin Rheumatol. 1999, 18: 346-348. 10.1007/s100670050115.View ArticleGoogle Scholar
  22. Rubin RL: Enzyme-linked immunosorbent assay for antibodies to native DNA, histones and (H2A-H2B)-DNA. In Manual of Clinical Laboratory Immunology. Edited by: Rose NR, de Macario EC, Folds JD, Lane HC, Nakamura RM. 1997, Washington, DC: American Society for Microbiology, 935-941.Google Scholar
  23. Laemmli UK: Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685.View ArticleGoogle Scholar
  24. Linstedt AD, Hauri HP: Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa. Mol Biol Cell. 1993, 4: 679-693.View ArticleGoogle Scholar
  25. Sonnichsen B, Lowe M, Levine T, Jamsa E, Dirac-Svejstrup B, Warren G: A role for giantin in docking COPI vesicles to Golgi membranes. J Cell Biol. 1998, 140: 1013-1021. 10.1083/jcb.140.5.1013.View ArticleGoogle Scholar
  26. Infante C, Ramos-Morales F, Fedriani C, Bornens M, Rios RM: GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein. J Cell Biol. 1999, 145: 83-98. 10.1083/jcb.145.1.83.View ArticleGoogle Scholar
  27. Bascom RA, Srinivasan S, Nussbaum RL: Identification and characterization of Golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain. J Biol Chem. 1999, 274: 2953-2962. 10.1074/jbc.274.5.2953.View ArticleGoogle Scholar
  28. Godi A, Santone I, Pertile P, Devarajan P, Stabach PR, Morrow JS, Di Tullio G, Polishchuk R, Petrucci TC, Luini A, De Matteis MA: ADP ribosylation factor regulates spectrin binding to the Golgi complex. Proc Natl Acad Sci USA. 1998, 95: 8607-8612. 10.1073/pnas.95.15.8607.View ArticleGoogle Scholar
  29. Fritzler MJ, Chan EKL: Autoantibodies to endosomes and the golgi complex. In Autoantigens and Autoantibodies: Diagnostic Tools and Clues to Understanding Autoimmunity. 2000, Lengerich, Germany: Pabst Scientific Publishers, 339-361.Google Scholar
  30. Selak S, Chan EKL, Schoenroth L, Senécal JL, Fritzler MJ: Early endosome antigen. 1: An autoantigen associated with neurological diseases. J Investig Med. 1999, 47: 311-318.Google Scholar
  31. Griffith KJ, Ryan JP, Senécal JL, Fritzler MJ: The cytoplasmic linker protein CLIP-170 is a human autoantigen. Clin Exp Immunol. 2002, 127: 533-538. 10.1046/j.1365-2249.2002.01756.x.View ArticleGoogle Scholar
  32. Doxsey SJ, Stein P, Evans L, Calarco PD, Kirschner M: Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell. 1994, 76: 639-650. 10.1016/0092-8674(94)90504-5.View ArticleGoogle Scholar
  33. Bouckson-Castaing V, Moudjou M, Ferguson DJ, Mucklow S, Belkaid Y, Milon G, Crocker PR: Molecular characterisation of ninein, a new coiled-coil protein of the centrosome. J Cell Sci. 1996, 109: 179-190.Google Scholar
  34. Mack GJ, Rees J, Sandblom O, Balczon R, Fritzler MJ, Rattner JB: Autoantibodies to a group of centrosomal proteins in human autoimmune sera reactive with the centrosome. Arthritis Rheum. 1998, 41: 551-558. 10.1002/1529-0131(199803)41:3<551::AID-ART22>3.3.CO;2-O.View ArticleGoogle Scholar
  35. Price CM, McCarty GA, Pettijohn DE: NuMA protein is a human autoantigen. Arthritis Rheum. 1984, 27: 774-779.View ArticleGoogle Scholar
  36. Andrade LEC, Chan EKL, Peebles CL, Tan EM: Two major autoantigen-antibody systems of the mitotic spindle apparatus. Arthritis Rheum. 1996, 39: 1643-1653.View ArticleGoogle Scholar
  37. Rattner JB, Rees J, Arnett FC, Reveille JD, Goldstein R, Fritzler MJ: The centromere kinesin-like protein, CENP-E. An autoantigen in systemic sclerosis. Arthritis Rheum. 1996, 39: 1355-1361.View ArticleGoogle Scholar
  38. Rattner JB, Rees J, Whitehead CM, Casiano CA, Tan EM, Humbel RL, Conrad K, Fritzler MJ: High frequency of neoplasia in patients with autoantibodies to centromere protein CENP-F. Clin Invest Med. 1997, 20: 308-319.Google Scholar
  39. Casiano CA, Ochs RL, Tan EM: Distinct cleavage products of nuclear proteins in apoptosis and necrosis revealed by autoantibody probes. Cell Death Differ. 1998, 5: 183-190. 10.1038/sj.cdd.4400336.View ArticleGoogle Scholar
  40. Casciola-Rosen L, Andrade F, Ulanet D, Wong WB, Rosen A: Cleavage by granzyme B is strongly predictive of autoantigen status: implications for initiation of autoimmunity. J Exp Med. 1999, 190: 815-826. 10.1084/jem.190.6.815.View ArticleGoogle Scholar
  41. Mancini M, Machamer CE, Roy S, Nicholson DW, Thornberry NA, Casciola-Rosen LA, Rosen A: Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis. J Cell Biol. 2000, 149: 603-612. 10.1083/jcb.149.3.603.View ArticleGoogle Scholar
  42. Casciola-Rosen LA, Anhalt G, Rosen A: Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med. 1994, 179: 1317-1330. 10.1084/jem.179.4.1317.View ArticleGoogle Scholar
  43. Lupas A, Van Dyke M, Stock J: Predicting coiled coils from protein sequences. Science. 1991, 252: 1162-1164.View ArticleGoogle Scholar


© Nozawa et al., licensee BioMed Central Ltd. 2004

This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.