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IMMUNOBIOLOGY
From the Department of Pathology, Faculty of Medicine,
University of Geneva, Switzerland; Institute of Glycotechnology and
Department of Applied Biochemistry, Tokai University, Hiratsuka,
Kanagawa, Japan; and Institut National de la Santé et de la
Recherche Médicale U 399, Marseille, France.
Autoantibodies of the cryoprecipitating IgG3 isotype have
been shown to play a significant role in the development of murine lupus-like autoimmune syndrome. At present, the structural basis of
IgG3 cryoprecipitation and its role in autoantibody pathogenicity remain to be defined. Using molecular variants of an IgG3 monoclonal rheumatoid factor, 6-19, derived from an autoimmune
MRL-Faslpr mouse, we have investigated the
implication of charged residues in the heavy-chain variable (VH)
region, potential CH3-linked oligosaccharides, and galactosylation of
CH2-linked oligosaccharides in its cryoglobulin activity. The
cryoglobulin activity of the IgG3 6-19 mutant bearing more negatively
charged residues at VH 6 and 23 was found to be reduced but still
highly significant, whereas that of the mutant lacking a potential CH3
glycosylation site remained unchanged. In marked contrast, IgG3 6-19 variants obtained from 6-19 heavy-chain transgenic mice displayed
barely detectable cryoglobulin activity associated with an increased level of galactosylation in the CH2 oligosaccharide side chains. Thus,
our data strongly suggest that the cryoglobulin activity of IgG3 6-19 autoantibody is critically determined by levels of galactosylation in
the CH2 oligosaccharide side chains, whereas VH residues play a
secondary role in 6-19 IgG3 cryoglobulin activity.
(Blood. 2002;99:2922-2928) Cryoglobulins are a heterogeneous group of
immunoglobulins (Ig) which precipitate upon cooling and redissolve on
warming. Most cryoglobulins are either intact monoclonal Ig or Ig
complexes in which one component, usually IgM, has rheumatoid factor
activity.1,2 Monoclonal cryoglobulins are mainly
associated with various lymphoproliferative disorders, whereas mixed
cryoglobulins are often found in serum from patients with autoimmune
diseases such as systemic lupus erythematosus and rheumatoid arthritis
or with chronic infectious diseases. The presence of cryoglobulins can
result in a wide range of vascular, renal, and neurologic
complications, probably depending on their concentrations, their
temperature-dependent solubility behavior, and the nature and type of
proteins involved.2 However, the precise pathogenic
mechanisms involved in the induction of these tissue lesions have not
been well defined.
In humans and mice, antibodies of the IgG3 isotype have a unique
physicochemical property, which allows them to self-associate via Fc-Fc
interactions, independently of their specificities.3-5 This property is necessary, but not sufficient in itself to confer cryoglobulin activity, since not all monoclonal IgG3 proteins in
humans6-8 and mice5,9,10 exhibit cyroglobulin
activity. However, the molecular basis responsible for the cryoglobulin formation by IgG3 molecules is still unclear. A study comparing nucleotide sequences of the variable (V) regions of cryogenic and
noncryogenic IgG3 monoclonal antibodies (mAbs) has claimed a role of
framework residues 6 and 23 of the heavy-chain V (VH) domain in IgG3
cryoprecipitation. These residues were more positively charged in
cryogenic IgG3 than in their noncryogenic counterparts.11 In addition, it has been suggested that oligosaccharide chains potentially present in the CH3 domain of murine IgG3, because of an
additional asparagine-linked glycosylation site in this domain,12 might be involved in the cryoglobulin activity
by promoting self-association of IgG3.13 Moreover, there
is remarkable heterogeneity in galactosylation levels of carbohydrate
side chains attached at residue 297 (asparagine) on the CH2 domain of
IgG,14 and these structural differences may play an
additional role for the cryoglobulin activity of self-associating IgG3
complexes. Most biantennary oligosaccharide chains are nonsialylated,
and end with either 2 terminal galactose residues, 1 terminal galactose and 1 terminal N-acetylglucosamine, or 2 terminal
N-acetylglucosamines.14 It has been shown that
the level of terminal galactosylation is substantially diminished in
some diseases,14-16 although it is still unknown whether
this association has a pathogenic significance.
We have previously established that an IgG3 anti-IgG2a
cryoglobulin derived from an autoimmune
MRL-Faslpr mouse is able to induce lupuslike
glomerulonephritis and skin vasculitis.10,17 Using this
model, we aimed to define the respective roles of VH residues 6 and 23, of potential CH3 oligosaccharides, and of galactosylation levels of CH2
oligosaccharide side chains in cryoglobulin activity of IgG3 mAb.
Therefore, we first generated 2 different 6-19 mutant rheumatoid factor
(RF) mAbs either by replacing VH residues 6 and 23 with those commonly
found in noncryogenic IgG3 mAbs, or by eliminating the potential CH3
glycosylation site, and compared their cryoglobulin activity with that
of the wild-type 6-19 RF mAb. Second, 3 "6-19" IgG3 RF mAbs were
established from mice bearing a transgene encoding the 6-19 IgG3 heavy
chain, which spontaneously produce IgG3 "6-19" mAbs by combining
the transgenic 6-19 IgG3 heavy chains with endogenous light chains. We
expected that these "6-19" mAbs, identical in the amino-acid
sequences of their heavy and light chains to those of the original 6-19 mAb, bore potentially different galactosylation patterns because of
their synthesis in different cells, as shown previously.18 Thus, we should be able to study the role of galactosylation in the CH2
oligosaccharides for the cryogenic potential of the 6-19 mAb. The
analysis of these 6-19 IgG3 RF variants revealed that the cryoglobulin
activity of the 6-19 mAb was primarily determined by the galactose
content of CH2 oligosaccharide chains, with a secondary involvement of
VH residues 6 and 23.
Monoclonal antibodies
Oligonucleotide-directed mutagenesis
RT-PCR and cDNA sequencing RNA was prepared from hybridomas by RNeasy Mini Kit (Qiagen AG, Basel, Switzerland). The first strand of cDNA was synthesized with an oligo(dT) primer and total RNA. For amplification with Pfu DNA polymerase (Stratagene Cloning Systems, La Jolla, CA), the following pairs of oligonucleotides were used: VH1BACK primer (5'-AGGTSMARCTGCAGSAGTCWGG-3')28 and C 3-CH1 primer
(5'-CGATAGACAGATGG-3') for the IgG3 VH region, 3'-untranslated 6-19 VH
primers (5'-CACTGACTTTCACCATG-3') and 3'-untranslated C3 primer
(5'-GACCCGAGGAATGGC-3') for the 6-19 IgG3 heavy-chain, and
Vk6-19 leader primer (5'-GTTGCCTCCTCAAATG-3') and
Ck primer (5'-TGGATGGTGGGAAGATG-3') for the light chain. The nucleotide sequence was determined by the dideoxynucleotide chain termination method.29
Enzyme-linked immunosorbent assay The 6-19 anti-IgG2a RF and 6-19 idiotype activities in IgG3 mAbs were determined by enzyme-linked immunosorbent assay (ELISA) as described.27,30 Concentrations of IgG3 in culture supernatants, sera, and cryoglobulins were determined by IgG3-specific ELISA as described.30Cryoglobulin activity A quantity of 0.1 mL purified IgG mAbs or sera was placed in conical glass tubes at 4°C for 48 hours. The samples were centrifuged at 3000 rpm at 4°C for 30 minutes. The precipitates were washed 3 times with cold phosphate-buffered saline (PBS) and resolubilized in 4 M urea. The concentrations of IgG in cryoprecipitates were determined by ELISA.IgG3 self-association assay The ability of the 6-19 mAb and its variants to form IgG3-IgG3 self-associating complexes was determined by a radioimmunoassay as described previously.10 Briefly, various amounts of mAbs were incubated with 125I-labeled 6-19 mAb (10 ng) at 4°C overnight in the presence of 10 µL normal mouse serum. After 1 hour of incubation at 4°C with 1 mL of 7.5% polyethylene glycol (Mr 6000; Merck, Dietikon, Switzerland), IgG3 complexes formed with radiolabeled 6-19 mAb were precipitated by centrifugation at 3000 rpm at 4°C for 30 minutes. The precipitates were washed once with 7.5% polyethylene glycol. Results are expressed as a percentage of labeled 6-19 mAb precipitated specifically after correction of the nonspecific precipitation (< 10%) obtained in the presence of normal mouse serum alone.Analysis of oligosaccharide structures The analysis of the structure of carbohydrate side chains present in the IgG3 samples was carried out as previously described.18 Briefly, IgG3 mAbs were purified from cultured supernatants by protein A column chromatography, followed by fast-performance liquid chromatography (FPLC) using a Superdex 200 pg column (Pharmacia, Uppsala, Sweden). The purified IgG3 mAbs were then subjected to gas-phase hydrazinolysis for 3 hours at 90°C using Hydraclub S204 (Honen, Tokyo, Japan) followed by N-acetylation to quantitatively liberate N-linked oligosaccharides.31 The oligosaccharides liberated from the IgG3 samples were purified by cellulose column chromatography32 and then labeled with P-aminobenzoic acid ethyl ester (ABEE; Nacalai Tesque, Kyoto, Japan) by reductive amination.33 The ABEE-labeled oligosaccharides were subjected to high-performance liquid chromatography (HPLC) using a COSMOGEL diethylaminoethyl column (Nacalai Tesque), and separated into neutral, monosialo, and disialo oligosaccharide fractions. Neutral oligosaccharide mixtures obtained by sialidase treatment of ABEE-oligosaccharide fraction were applied to HPLC using a Wakosil 5C18-200 octadecylsilane column (Wako Pure Chemical Industries, Kyoto, Japan). Desialylated ABEE-oligosaccharides were separated into 8 oligosaccharide fractions which were eluted as a series of authentic biantennary complex-type oligosaccharides with or without fucosylation.
VH residues 6 and 23 influence but do not critically determine the cryoglobulin activity of murine IgG3 It has been reported that neutral glutamine and cationic lysine are found at VH positions 6 and 23, respectively, in 6 murine IgG3 cryoglobulins, including the 6-19 RF mAb, whereas their counterparts in 5 noncryoglobulins are more negatively charged at these residues: anionic glutamic acid at position 6 and neutral amino acid (alanine, threonine, or valine) at position 23.11 To specifically assess the influence of the VH residues 6 and 23 on the IgG3 cryoglobulin activity, we determined the effect of mutations of these 2 residues on the cryogenic potential of the 6-19 IgG3 mAb. A comparative analysis with the wild-type 6-19 mAb showed that the cryoglobulin activity of the 6-19Q6E/K23A mutant bearing more negatively charged residues at VH positions 6 (glutamic acid instead of glutamine) and 23 (alanine instead of lysine) was approximately 2 times lower than that of the wild-type 6-19 mAb, but still highly significant (Table 1). Notably, anti-IgG2a RF activity of the 6-19Q6E/K23A mutant was comparable with that of the wild-type 6-19 RF mAb (Figure 1).
To extend this observation, we carried out analysis of an additional 8 cryoprecipitating and 6 noncryoprecipitating IgG3 mAbs. Although the VH
residues 6 and 23 are in general more positively charged in the
cryoprecipitating populations, this is not always the case (Table
2). DP7 anti-DNA, S57 anti-DNA, and
411H12 anti-IgG1 mAbs bearing more positively charged VH residues 6 (glutamine) and 23 (lysine) like cryogenic IgG3 fail to
cryoprecipitate. To the contrary, SH11 anti-DNA and C11M anti-DNP mAbs
with residues like noncryoprecipitating IgG3 exhibit a cryoglobulin
activity.
Self-association and cryoglobulin activity of murine IgG3 is independent of potential oligosaccharide chains on the CH3 domain A unique feature of the murine IgG3 isotype is the presence of a potential asparagine-linked glycosylation site at residues 471-473 (asparagine-leucine-serine) in the CH3 domain.12 Although it is unknown whether murine IgG3 is indeed glycosylated in the CH3 domain, CH3 oligosaccharide chains, if present, might play a role in the cryoglobulin activity of murine IgG3 by modifying its self-associating property, as suggested by Panka.13 To verify this possibility, we generated a 6-19N471T mutant mAb, in which asparagine at position 471 was replaced by threonine, thus eliminating the potential CH3 N-linked glycosylation site. When various amounts of 6-19 or 6-19N471T mutant mAb were incubated with radiolabeled 6-19 protein, both mAbs exhibited a concentration-dependent formation of IgG3-IgG3 complexes in a quantitatively identical manner (Figure 2). In addition, the ability of the 6-19N471T mutant to generate cryoprecipitates following a 48-hour incubation at 4°C was indistinguishable from that of the wild-type 6-19 mAb (Table 1). In contrast, no significant IgG3 complex and cryoglobulin formation was observed with the 6-19 IgG1 switch variant, SS2F8 (Figure 2 and Table 1). Significantly, the content of N-linked oligosaccharides liberated from the 6-19N471T mutant was essentially identical to that of the wild-type 6-19 and 6-19Q6E/K23A mutant, which was not higher than that of its IgG1 variant, SS2F8, (Table 1), strongly arguing against the possible glycosylation on the IgG3 CH3 domain.
Lack of cryoglobulin activity of 6-19 somatic variants derived from 6-19 heavy-chain transgenic mice The extent of the terminal galactosylation of oligosaccharide side chains on the IgG CH2 domain is highly variable14-16 and depends on the cellular environment.34 We therefore explored the possibility that different levels of galactosylation influence the cryoprecipitating potential of IgG3 self-associating complexes, using 6-19 variants identical in the amino-acid sequences of their heavy and light chains, but different in galactosylation patterns because of their synthesis in different hybridoma cells. To obtain such variants, a series of IgG3 anti-IgG2a RF mAbs expressing the 6-19 idiotype were generated from 6-19 heavy-chain transgenic mice, which spontaneously produce substantial amounts of 6-19-like IgG3 anti-IgG2a RF, resulting from the combination of the transgenic 6-19 heavy chains with endogenous light chains. The 6-19 RF mAb, derived from MRL-Faslpr mice (Ighj), utilizes the Jk2' gene segment,27 an Ighj-linked allelic form of the Jk2 gene. Therefore, 6-19 heavy-chain transgenic mice were crossed once with MRL mice to introduce the Ighj allotype. Using their spleen cells, 12 IgG3 anti-IgG2a RF mAbs bearing the 6-19 idiotype were established. Nucleotide sequence analysis of light-chain cDNA revealed that all 6-19-like mAbs utilize the Vk1c germ-line gene, identical to that of the 6-19 mAb,27 but different Jk segments (Table 3). Among them, light chains of 3 mAbs (A6C, P11C, and Y12C), established from 3 independent fusions, use the Jk2' segment identical to that of the 6-19 mAb.
Using these 3 6-19 derived variants, A6C, P11C, and Y12C, cryoglobulin
activity was assessed in vitro following a 48 hour incubation at 4°C.
Strikingly, none of the A6C, P11C, and Y12C mAbs showed evidence for
cryoprecipitation (Figure 3A). Superdex 200HR gel filtration chromatography and SDS-PAGE analysis excluded the
possibility that the lack of cryoglobulin activity by A6C, P11C, and
Y12C mAbs was due to the formation of an abnormal truncated form or an
aberrant assembly of their heavy and light chains in the hybridoma
cells (data not shown). Notably, nucleotide sequence analysis of their
heavy-chain cDNA confirmed that these 3 mAbs bear heavy chains
identical to that of the 6-19 RF mAb, consistent with the fact that
their anti-IgG2a RF activities were comparable with that of the 6-19 mAb (Figure 3B). Noteworthy, the self-associating capacities of these 3 mAbs were indistinguishable from that of the 6-19 mAb (Figure 3C).
Different levels of galactosylation in the cryoprecipitating 6-19 mAb and noncryoprecipitating 6-19 somatic variants To determine whether the lack of the cryoglobulin activity of the 3 6-19 variants, A6C, P11C, and Y12C, can be attributed to a possible structural difference in the carbohydrate side chains present in the CH2 domain, the oligosaccharide structures liberated from these 3 mAbs were compared with those of the cryoprecipitating 6-19, 6-19Q6E/K23A, and 6-19N471T mAbs. Analysis by diethylaminoethyl-HPLC showed comparable molar ratios of neutral asialo-, acidic monosialo-, and acidic disialo-oligosaccharides, in which neutral nonsialylated oligosaccharides amounted to approximately 90% of total oligosaccharides (Table 4).
Octadecylsilane-HPLC analysis of the neutral oligosaccharides obtained
after sialidase treatment of total oligosaccharides showed no
measurable differences in the level of fucosylation among the different
6-19 mAbs (Figure 4), fucosylated
oligosaccharides amounting to more than 95% of neutral
oligosaccharides. In contrast, cryogenic and noncryogenic 6-19 mAbs
differed in galactosylation patterns. The content of nongalactosylated
(G0; no galactose, terminating in N-acetylglucosamine)
oligosaccharides was uniformly elevated in the cryogenic 6-19, 6-19Q6E/K23A, and 6-19N471T mAbs, as compared with the noncryogenic
A6C, P11C, and Y12C mAbs (peak 8 in Figure 4 and Table 4). Moreover,
the cryogenic 6-19 mAbs had approximately 2-fold lower content of
digalactosylated (G2; 2 terminal galactose residues) oligosaccharides
than the noncryogenic 6-19 variants (peak 5 in Figure 4 and Table 4).
Consequently, the ratio of the nongalactosylated (G0) versus
galactosylated (G1 + G2) oligosaccharides was 2 times higher in
the cryoprecipitating 6-19 mAbs than in the noncryoprecipitating 6-19 variants.
The present study was designed to investigate the molecular basis of the cryogenic potential of murine IgG3 by using several different variants of an 6-19 IgG3 RF mAb. Our analysis has demonstrated that the cryoglobulin activity of the 6-19 mAb was reduced, but still highly significant in its Q6E/K23A mutant, whereas it was hardly detectable in its somatic variants bearing an increased content of galactose residues in the oligosaccharide side chains present in the CH2 domain of their heavy chains. Our results indicate that the level of IgG galactosylation critically determines the IgG3 cryoglobulin activity, with a secondary role of VH residues. A previous analysis of a panel of IgG3 mAbs (6 cryoglobulins and 5 noncryoglobulins) has shown that cryogenic IgG3 mAbs are more positively charged at VH residues 6 and 23 than noncryogenic IgG3 mAbs.11 However, the present analysis of 14 additional IgG3 mAbs failed to show a complete correlation of the presence of more positive residues at VH 6 and 23 with the cryoglobulin activity. Furthermore, we have observed that the 6-19Q6E/K23A mutant mAb, VH residues 6 and 23 of which were mutated to noncryoglobulinlike residues, still exhibited significant cryoglobulin activity. Strikingly, A6C, P11C, and Y12C mAbs, identical in the amino-acid sequences of their heavy and light chains to those of the 6-19 mAb, were found to be hardly cryoprecipitable. These results clearly indicate that VH residues 6 and 23 are not the only factor determining the cryoglobulin activity of murine IgG3. Most significantly, comparative analysis of the oligosaccharide
structures of different 6-19 mAbs revealed that the level of
galactosylation was substantially decreased in the cryogenic 6-19 mAbs,
as compared with those of the noncryogenic 6-19 variants, A6C, P11C,
and Y12C. Thus, the galactose content of CH2 oligosaccharide side
chains plays a key role in determining the cryoglobulin activity of the
6-19 IgG3 mAb. At present, we lack a good explanation for the role of
galactosylation in cryoglobulin activity of murine IgG3 mAbs. The
terminal galactose residue apparently forms a tight interaction with a
lectinlike binding pocket on the CH2 domain of IgG, which partly plays
a role in maintaining the relative geometry of the 2 CH2
domains.35,36 Thus, the absence of the terminal galactose
could lead to alterations in the conformation of IgG3 molecules,
thereby promoting their cryoprecipitating potential. It should,
however, be mentioned that we have recently observed that the 6-19J mAb
bearing the 6-19 heavy chains and J558 Because of the presence of an additional potential N-linked glycosylation site in the CH3 domain of murine IgG3 isotype,12 it has been speculated that possible CH3 oligosaccharide chains may play a role in the self-associating property of murine IgG3 and hence cryoglobulin activity.13 However, this possibility is excluded for 2 reasons. First, the 6-19N471T mutant mAb lacking this potential glycosylation site is able to self-associate and generate cryoglobulins as efficiently as the wild-type 6-19 mAb. Second, the content of N-linked oligosaccharides of the 6-19N471T mutant was not different from that of the wild-type 6-19 mAb as well as that of its IgG1 variant mAb lacking the potential to self-associate and cryoprecipitate. Collectively, this indicates the absence of glycosylation at the CH3 domain of murine IgG3. The present results contrast with those described by Panka, who claimed that the self-associating ability of IgG3 was significantly reduced in a similar N-linked glycosylation site-loss mutant.13 This discrepancy could be due to the assay used by Panka's study, in which the self-associating activity was measured by a multistep, solid-phase absorbent assay, instead of a direct binding assay in a liquid-phase system, as originally proposed5 and used in the present and previous studies.10,11 It should be also mentioned that asparagine at position 471 was replaced by threonine in our mutant, instead of serine in the study by Panka. Thus, we cannot totally exclude the possibility that the change of asparagine to serine, but not threonine, may affect the self-associating property of murine IgG3. Our demonstration that a decrease in the relative content of galactose residues in the CH2 oligosaccharide chains is associated with IgG3 cryoglobulin activity suggests that the level of IgG galactosylation may be important in determining the pathogenic potential of autoantibodies with cryoglobulin activity. We observed the development of cryoglobulinemia associated with glomerulonephritis and skin leukocytoclastic vasculitis in mice implanted with transfectoma cells secreting 6-19Q6E/K23A mutant mAbs, as in the case of 6-19 hybridoma-injected mice, whereas no lesions were induced in mice transplanted with hybridoma cells secreting the 6-19 variants, A6C, P11C, or Y12C, lacking cryoglobulin activity (A.K., S.K., Y.K., et al, unpublished data, March 2001). However, the interpretation of these results was difficult, because serum levels of IgG3 concentrations in the latter group of mice have never exceeded more than 1 mg/mL, in contrast with the concentrations (4 mg/mL-5 mg/mL) obtained with mice injected with 6-19 hybridoma or 6-19Q6E/K23A transfectoma cells. In fact, the capacity of A6C, P11C, and Y12C hybridoma cells to secrete IgG3 mAbs was approximately 5 to 10 times less than that of the 6-19 hybridoma or 6-19Q6E/K23A transfectoma cells. The experiment with a hybridoma secreting sufficient amounts of IgG3 "6-19" mAbs lacking cryoglobulin activity could define the role of cryoglobulin activity in the pathogenic potential of IgG3 monoclonal cryoglobulins. Clearly, further elucidation of the pathogenic role of cryoglobulin activity and decreased IgG galactosylation would help develop new diagnostic and therapeutic approaches for cryoglobulin- and autoantibody-mediated disorders.
We thank Mr T. Moll for critically reading the manuscript, and Ms G. Leyvraz, Mr G. Brighouse, Mr G. Celetta, and Ms P. Henchoz for their excellent technical help.
Submitted September 28, 2001; accepted December 6, 2001.
Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture and from the Ministry of Health and Welfare of Japan, Tokai University, and the Swiss National Foundation for Scientific Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Shozo Izui, Department of Pathology, CMU, 1211 Geneva 4, Switzerland; e-mail: shozo.izui{at}medecine.unige.ch.
1. Grey HM, Kohler PF. Cryoimmunoglobulins. Semin Hematol. 1973;10:87-112[Medline] [Order article via Infotrieve]. 2. Brouet JC, Clauvel JP, Danon F, Klein M, Seligmann M. Biological and clinical significance of cryoglobulins: a report of 86 cases. Am J Med. 1974;57:775-788[CrossRef][Medline] [Order article via Infotrieve].
3.
Capra JD, Kunkel HG.
Aggregation of 4. Grey HM, Hirst JW, Cohn M. A new mouse immunoglobulin: IgG3. J Exp Med. 1971;133:289-304[Abstract]. 5. Abdelmoula M, Spertini F, Shibata T, et al. IgG3 is the major source of cryoglobulins in mice. J Immunol. 1989;143:526-532[Abstract].
6.
Grey HM, Kohler PF, Terry WD, Franklin EC.
Human monoclonal 7. Saluk PH, Clem W. Studies on the cryoprecipitation of a human IgG3 cryoglobulin: the effects of temperature-induced conformational changes on the primary interaction. Immunochemistry. 1975;12:29-37[CrossRef][Medline] [Order article via Infotrieve].
8.
Nishimura Y, Nakamura H.
Human monoclonal cryoimmunoglobulins, I: molecular properties of IgG3k (Jir protein) and the cryo-coprecipitability of its molecular fargments by papain.
J Biochem.
1984;95:255-265 9. Spertini F, Coulie PG, Van Snick J, Davidson E, Lambert P-H, Izui S. Inhibition of cryoprecipitation of murine IgG3 anti-dinitrophenyl (DNP) monoclonal antibodies by anionic DNP-amino acid conjugates. Eur J Immunol. 1989;19:273-278[Medline] [Order article via Infotrieve].
10.
Fulpius T, Spertini F, Reininger L, Izui S.
Immunoglobulin heavy chain constant region determines the pathogenicity and the antigen-binding activity of rheumatoid factor.
Proc Natl Acad Sci U S A.
1993;90:2345-2349 11. Panka DJ, Salant DJ, Jacobson BA, Minto AW, Marshak-Rothstein A. The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition. Eur J Immunol. 1995;25:279-284[Medline] [Order article via Infotrieve]. 12. Wels JA, Word CJ, Rimm D, et al. Structural analysis of the murine IgG3 constant region gene. EMBO J. 1984;3:2041-2046[Medline] [Order article via Infotrieve]. 13. Panka DJ. Glycosylation is influential in murine IgG3 self-association. Mol Immunol. 1997;34:593-598[CrossRef][Medline] [Order article via Infotrieve]. 14. Parekh RB, Dwek RA, Sutton BJ, et al. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature. 1985;316:452-457[CrossRef][Medline] [Order article via Infotrieve]. 15. Parekh R, Isenberg D, Rook G, Roitt I, Dwek R, Rademacher T. A comparative analysis of disease-associated changes in the glycosylation of serum IgG. J Autoimm. 1989;2:101-114. 16. Mizuochi T, Hamako J, Nose M, Titani K. Structural changes in the oligosaccharide chains of IgG in autoimmune MRL/Mp-lpr/lpr mice. J Immunol. 1990;145:1794-1798[Abstract]. 17. Gyotoku Y, Abdelmoula M, Spertini F, Izui S, Lambert PH. Cryoglobulinemia induced by monoclonal immunoglobulin G rheumatoid factors derived from autoimmune MRL/MpJ-lpr/lpr mice. J Immunol. 1987;138:3785-3792[Abstract].
18.
Mizuochi T, Pastore Y, Shikata K, et al.
Role of galactosylation in the renal pathogenicity of murine IgG3 monoclonal cryoglobulins.
Blood.
2001;97:3537-3543
19.
Shlomchik M, Mascelli M, Shan H, et al.
Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation.
J Exp Med.
1990;171:265-292 20. Shlomchik MJ, Marshak-Rothstein A, Wolfowicz CB, Rothstein TL, Weigert MG. The role of clonal selection and somatic mutation in autoimmunity. Nature. 1987;328:805-811[CrossRef][Medline] [Order article via Infotrieve]. 21. Takahashi S, Itoh J, Nose M, Ono M, Yamamoto T, Kyogoku M. Cloning and cDNA sequence analysis of nephritogenic monoclonal antibodies derived from an MRL/lpr lupus mouse. Mol Immunol. 1993;30:177-182[CrossRef][Medline] [Order article via Infotrieve]. 22. Jovelin F, Mostoslavsky G, Amoura Z, et al. Early anti-nucleosome autoantibodies from a single MRL +/+ mouse: fine specificity, V gene structure and pathogenicity. Eur J Immunol. 1998;28:3411-3422[CrossRef][Medline] [Order article via Infotrieve].
23.
Shefner R, Kleiner G, Turken A, Papazian L, Diamond B.
A novel class of anti-DNA antibodies identified in BALB/c mice.
J Exp Med.
1991;173:287-296 24. Swanson PC, Yung RL, Blatt NB, et al. Ligand recognition by murine anti-DNA autoantibodies. J Clin Invest. 1996;97:1748-1760[Medline] [Order article via Infotrieve]. 25. Berney T, Shibata T, Izui S. Murine cryoglobulinemia: pathogenic and protective IgG3 self-associating antibodies. J Immunol. 1991;147:3331-3335[Abstract]. 26. Lemoine R, Berney T, Shibata T, et al. Induction of "wire-loop" lesions by murine monoclonal IgG3 cryoglobulins. Kidney Int. 1992;41:65-72[Medline] [Order article via Infotrieve].
27.
Reininger L, Berney T, Shibata T, Spertini F, Merino R, Izui S.
Cryoglobulinemia induced by a murine IgG3 rheumatoid factor: skin vasculitis and glomerulonephritis arise from distinct pathogenic mechanisms.
Proc Natl Acad Sci U S A.
1990;87:10038-10042
28.
Orlandi R, Güssow DH, Jones PT, Winter G.
Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.
Proc Natl Acad Sci U S A.
1989;86:3833-3837
29.
Sanger FS, Nicklen S, Coulson AR.
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci U S A.
1977;74:5463-5467
30.
Berney T, Fulpius T, Shibata T, et al.
Selective pathogenicity of murine rheumatoid factors of the cryoprecipitable IgG3 subclass.
Int Immunol.
1992;4:93-99 31. Mizuochi T. Microscale sequencing of N-linked oligosaccharides of glycoprotein using hydrolysis, Bio-Gel P-4, and sequential exoglycosidase. Methods Mol Biol. 1993;14:55-68[Medline] [Order article via Infotrieve]. 32. Shimizu Y, Nakata M, Kuroda Y, Tsutsumi F, Kojima N, Mizuochi T. Rapid and simple preparation of N-linked oligosaccharides by celluose column chromatography. Carbohydr Res. 2001;332:381-388[CrossRef][Medline] [Order article via Infotrieve]. 33. Shikata K, Yasuda T, Takeuchi F, Konishi T, Nakata M, Mizuochi T. Structural changes in the oligosaccharide moiety of human IgG with aging. Glycoconjugate J. 1998;15:683-689[CrossRef][Medline] [Order article via Infotrieve]. 34. Mizuochi T, Taniguchi T, Shimizu A, Kobata A. Structural and numerical variations of the carbohydrate moiety of immunoglobulin. J Immunol. 1982;129:2016-2020[Abstract].
35.
Lund J, Takahashi N, Pound JD, Goodall M, Nakagawa H, Jefferis R.
Oligosaccharide-protein interactions in IgG can modulate recognition by Fc
36.
Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA.
Glycosylation and the immune system.
Science.
2001;291:2370-2376
37.
Rengers JU, Touchard G, Decourt C, Deret S, Michel H, Cogné M.
Heavy and light chain primary structures control IgG3 nephritogenicity in an experimental model for cryocrystalglobulinemia.
Blood.
2000;95:3467-3472
© 2002 by The American Society of Hematology.
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  L. Baudino, F. Nimmerjahn, S. Azeredo da Silveira, E. Martinez-Soria, T. Saito, M. Carroll, J. V. Ravetch, J. S. Verbeek, and S. Izui Differential Contribution of Three Activating IgG Fc Receptors (Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV) to IgG2a- and IgG2b-Induced Autoimmune Hemolytic Anemia in Mice J. Immunol., February 1, 2008; |
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Abstract
Introduction
) and 6-19Q6E/K23A (
)
mAb was determined by ELISA, and results are expressed as OD at
405 nm.
), were incubated with 125I-labeled 6-19 mAb (10 ng) in the presence of 10 µL normal mouse serum. Results are
expressed as a percentage of labeled 6-19 precipitated
specifically.
), and Y12C (
) mAbs were incubated for 48 hours at 4°C.
Results are expressed concentrations of IgG3 in cryoprecipitates. Note
the lack of cryoprecipitation of A6C, P11C, and Y12C mAbs, in contrast
to the generation of significant amounts of cryoglobulins by the 6-19 mAb even at a concentration of 0.25 mg/mL. (B) Anti-IgG2a RF activity
of purified 6-19 (
1-4GlcNAc
1-6(±Gal
1 light chains was still
cryogenic despite an even higher galactose content than noncryogenic
6-19 variants.