A group A streptococcal Enn protein potentially resulting from intergenomic recombination exhibits atypical immunoglobulin-binding characteristics

The gene encoding the Enn protein (enn) of the M untypeable group A streptococcal (GAS) strain 64/14 was amplified by polymerase chain reaction, cloned into the expression vector pJLA602 and expressed in Escherichia coli DH5α. Unlike other GAS–Enn proteins, which exhibit IgA-binding activity, the recombinant Enn enn64/14 protein reacted preferentially with human IgG₃. The 1050 bp open reading frame comprising the enn64/14 gene was completely sequenced. The region of the gene encoding the signal peptide and the C-terminus exhibited >95% homology to corresponding sections of other enn genes. The region of enn64/74 encoding the N-terminus of the mature Enn protein was found to be highly homologous to the corresponding section of the gene encoding the M-like protein of GAS serotype M9 (emmL9). The reoombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152bp open reading frame of emmiL9 encoding the N-terminus was found to display >90% homology to the corresponding section of enn64/14 but was <50% homologous in the remainder of the gene sequence. The functional analysis of the subcloned N-terminal section of emmL9 demonstrated a polypeptide exhibiting selective binding to human IgG₃. These findings suggested that enn64/14 was a hybrid gene formed by recombination of an enn gene and an emmL9 gene. The putative recombinational event could have Involved a set of flanking 7bp direct repeats. Since enn64/14 and emmL9 are genes from different phylogenetic lineages of GAS, this report provides evidence that intergenomic recombinations between different types of GAS genes can occur and could lead to hybrid proteins with unique Ig-binding characteristics.     

Large-Scale In Vitro Expansion of Polyclonal Human Switched-Memory B Lymphocytes

Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients’ accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×10⁶-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG₁, IgG₂, IgG₃ and IgG₄ is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes.     

Relationship between Expression of the Family of M Proteins and Lipoteichoic Acid to Hydrophobicity and Biofilm Formation in Streptococcus pyogenes

Background

Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms.

Methodology/Principal Findings

Isogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family.

Conclusions/Significance

Our findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.     

The Interactions of Porcine and Ovine,Serum and Colostral Immunoglobulins with Staphylococcal Protein A

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produced by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG₁, IgG₂, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG₁ from most ewes possessed a low affinity for Protein A whereas ovine IgG₂ generally possessed a high affinity; 100% of the IgG₂ in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.     

Genetic variability of the emm-related genes of the large vir regulon of group A streptococci: potential intra- and intergenomic recombination events

One of the most prevalent genetic lineages of group A streptococci (GAS) harbors a genomic locus termed the large vir regulon, which contains an emm gene encoding the antiphagocytic M protein, and structurally related fcrA and enn (emm-related) genes encoding immunoglobulin-binding proteins. In the present study more than 100 large vir regulons from 42 different GAS serotypes were analyzed by PCR and partial DNA sequencing. On comparing these data to published sequences, sites of mutational and putative recombinational events were identified and ordered with respect to their intra/intergenic or intra/intergenomic nature. The emm-related genes were found to display small intragenic deletions or insertions, were completely deleted from, or newly inserted into the genome, or were fused to adjacent genes. Intergenomic exchanges of complete emm-related genes, or segments thereof, between different vir regulons were detected. Most of these processes seem to involve short flanking direct repeats. Occasionally, the structural changes could be correlated with changes in the functions of the encoded proteins.     

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein a and rabbit IgG

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A fromStaphylococcus aureus (termed SpA¹) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA¹ and IgG-Fc were examined by the individual substitution of the residues in SpA¹ involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA¹ proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbant assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG₁-Fc (Fc fragment of human IgG₁) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.     

Trypanosoma lewisi: antibody classes which mediate precipitation, agglutination, and the inhibition of reproduction

Sera from Trypanosoma lewisi-infected and uninfected rats were applied to Protein A-Sepharose CL-4B columns. The absorbed fractions of antisera which contained only IgG molecules were reacted in microimmunodiffusion analyses with the exoantigens of T. lewisi in plasma collected from irradiated infected rats, and formed one precipitin line. These sera were also applied to T. lewisi extract immunoabsorbent columns and bound proteins were eluted and analyzed by immunodiffusion against antisera specific for rat immunoglobulins. IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM were absorbed by the immuno-absorbent columns. Absorption of the rat antisera with anti-rat IgG or anti-rat IgM removed one of the two precipitin lines against extracts prepared from parasites collected from irradiated infected animals. The absorbed IgG fractions and nonabsorbed fractions of antisera which were collected after Protein A-Sepharose CL-4B column chromatography agglutinated trypanosomes. After treatment of antisera with 2-mercaptoethanol, the agglutinin titers were lower than those of the control antisera suggesting both IgG and IgM are involved in the agglutination. The ablastic activity of the fractions eluted from Protein A-Sepharose CL-4B Chromatographic columns was assayed in cultures of bloodstream forms ofT. lewisi. Ablastic activity of proteins of antisera absorbed by the columns was demonstrated indicating they belonged to the IgG class of antibodies.     

Molecular population genetic analysis of the enn subdivision of group A streptococcal emm-like genes: horizontal gene transfer and restricted variation among enn genes

The group A streptococcal emm-like genes, which encode the cell-surface M and M-like proteins, are divided into distinct mrp, emm and enn subdivisions and are clustered together in a region of the chromosome called the vir regulon. In order to understand the mechanisms involved in the evolution of emm-like genes, a 180bp fragment of the 5 variable region of the enn gene was characterized in 31 strains for which emm sequences and multilocus enzyme electrophoretic profiles have been previously determined. The results demonstrate that nucleotide polymorphisms at the enn locus are generated predominantly by point mutations and short deletions or insertions, and that variation among enn and emm genes has arisen by similar mechanisms. However, diversity at the enn locus is restricted in comparison to the emm locus. Moreover, there is strong evidence for intragenic recombination at the enn locus and the pattern of distribution of emm and enn alleles among strains suggests that these genes may be independently acquired by horizontal transfer and recombination from distinct donor strains, thereby generating a mosaic structure for the vir regulon. The results add to a growing body of evidence that horizontal gene transfer has played a major role in the evolution of Streptococcus pyogenes vir regulons.     

Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis

The novel species Halomonas zhaodongensis NEAU-ST10-25^T recently identified by our group is a moderate halophile which can grow at the range of 0–2.5 M NaCl (optimum 0.5 M) and pH 6–12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25^T in this study for Na⁺(Li⁺)/H⁺ antiporter genes by selection in Escherichia coli KNabc lacking three major Na⁺(Li⁺)/H⁺ antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.     

Reactivity of bacterial Fc receptors with bovine immunoglobulins: implications and limitations for quantitative immunochemistry

Summary The interaction of bovine immunoglobulins with staphylococcal Protein A and a group C streptococcal bacterial Fc receptor (FcRc) were compared. The isolated group C streptococcal receptor was reactive with both bovine IgG₁ and IgG₂. The reactivity of the streptococcal FcRc with IgG₂ was approximately 40 fold greater than that observed with IgG₁. By contrast, protein A reacted only poorly with bovine IgG₂ and no detectable reactivity was observed with IgG₁. A two stage competitive binding assay to measure bovine IgG in serum and secretions using ¹²⁵I-labeled protein A as tracer was developed. This assay was found to be sensitive and reproducible and was used to measure serum IgG levels in cattle of differing ages and breeds.     

Mesangial Cell-Binding Activity of Serum Immunoglobulin G in Patients with Lupus Nephritis

In vitro data showed that immunoglobulin G (IgG) from patients with lupus nephritis (LN) could bind to cultured human mesangial cells (HMC). The clinical relevance of such binding was unknown. Binding of IgG and subclasses was measured in 189 serial serum samples from 23 patients with Class III/IV±V LN (48 during renal flares, 141 during low level disease activity (LLDA)). 64 patients with non-lupus glomerular diseases (NLGD) and 23 healthy individuals were used as controls. HMC-binding was measured with cellular ELISA and expressed as OD index. HMC-binding index of total IgG was 0.12±0.09, 0.36±0.25, 0.59±0.37 and 0.74±0.42 in healthy subjects, NLGD, LN patients during LLDA, and LN flares respectively (P = 0.046, LN flare vs. LLDA; P<0.001, for healthy controls or NLGD vs. LN during flare or LLDA). Binding of serum IgG₁ to HMC was 0.05±0.05, 0.15±0.11, 0.41±0.38 and 0.55±0.40 for the corresponding groups respectively (P = 0.007, LN flare vs. remission; P<0.001, for healthy controls or NLGD vs. LN during flare or remission). IgG₂, IgG₃ and IgG₄ from patients and controls did not show significant binding to HMC. Total IgG and IgG₁ HMC-binding index correlated with anti-dsDNA level (r = 0.26 and 0.39 respectively, P<0.001 for both), and inversely with C3 (r = −0.17 and −0.45, P<0.05 for both). Sensitivity/specificity of total IgG or IgG₁ binding to HMC in predicting renal flares were 81.3%/39.7% (ROC AUC 0.61, P = 0.03) and 83.8%/41.8% (AUC 0.63, P = 0.009) respectively. HMC-binding by IgG_1, but not total IgG, correlated with mesangial immune deposition in LN renal biopsies under electron microscopy. Our results showed that binding of serum total IgG and IgG₁ in LN patients correlates with disease activity. The correlation between IgG₁ HMC-binding and mesangial immune deposition suggests a potential pathogenic significance.     

Seven-Year Surveillance of emm Types of Pediatric Group A Streptococcal Pharyngitis Isolates in Western Greece

Background

An experimental 26-valent M protein Group A streptococcal (GAS) vaccine has entered clinical studies. Pharyngeal GAS emm type surveillances in different areas and time-periods enhance the understanding of the epidemiology of GAS pharyngitis. Moreover, these surveillances, combined with the data on GAS invasive disease, can play a significant role in the formulation of multivalent type-specific vaccines.

Methods

During a 7-year period (1999–2005), 2408 GAS isolates were recovered from consecutive children with pharyngitis in Western Greece. The overall macrolide resistance rate was 22.8%. Along the study period we noted a tendency towards significantly decreased rates of resistance, with the lowest rates occurring in 2002 (15.3%), 2003 (15%) and 2004 (16.7%). A random sample of isolates from each year, 338 (61.7%) of the 548 macrolide-resistant and 205 (11%) of the macrolide-susceptible, underwent molecular analysis, including emm typing.

Results

The 543 typed isolates had 28 different emm types. A statistically significant association was found between macrolide resistance and emm4, emm22 and emm77, whereas emm1, emm3, emm6, emm12, emm87 and emm89 were associated with macrolide susceptibility. A significant yearly fluctuation was observed in emm4, emm28 and emm77. The most common macrolide-resistant GAS were emm77 isolates harboring erm(A), either alone or in combination with mef(A), emm4 carrying mef(A), emm28 possessing erm(B), emm75 carrying mef(A), emm12 harboring mef(A) and emm22 carrying erm(A). We estimated that 82.8% of the isolates belonged to emm types included in the novel 26-valent M protein vaccine. The vaccine coverage rate was determined mainly by the increased frequency of nonvaccine emm4 isolates.

Conclusions

A limited number of emm types dominated among macrolide-susceptible and macrolide-resistant GAS isolates. We observed seasonal fluctuations, which were significant for emm4, emm28 and emm77. This type of data can serve as baseline information if the novel 26-valent M protein GAS vaccine is introduced into practice.     

Genetic variability of the emm-related genes of the large vir regulon of group A streptococci: potential intra- and intergenomic recombination events

One of the most prevalent genetic lineages of group A streptococci (GAS) harbors a genomic locus termed the large vir regulon, which contains an emm gene encoding the antiphagocytic M protein, and structurally related fcrA and enn (emm-related) genes encoding immunoglobulin-binding proteins. In the present study more than 100 large vir regulons from 42 different GAS serotypes were analyzed by PCR and partial DNA sequencing. On comparing these data to published sequences, sites of mutational and putative recombinational events were identified and ordered with respect to their intra/intergenic or intra/intergenomic nature. The emm-related genes were found to display small intragenic deletions or insertions, were completely deleted from, or newly inserted into the genome, or were fused to adjacent genes. Intergenomic exchanges of complete emm-related genes, or segments thereof, between different vir regulons were detected. Most of these processes seem to involve short flanking direct repeats. Occasionally, the structural changes could be correlated with changes in the functions of the encoded proteins.     

Sequencing of genes within the vir regulon of Streptococcus pyogenes type M15 — an opacity factor-positive serotype with low opacity factor expression

Major virulence determinants of group A streptococci, such as M-protein, immunoglobulin Fc-receptors (FcRA, EmmL) and C5a peptidase, appear to be genetically co-regulated, their genes being located within a vir regulon. We studied the organization of these genes in a group A, type M15 strain of Streptococcus pyogenes, previously defined as OF^?, by hybridization analysis of chromosomal DNA and of an S. pyogenes gene library in Escherichia coli, and by gene sequencing. Within the vir regulon, in addition to the virR and scpA genes, three so-called emm-related genes were found: fcrA, emmL and enn. Whereas IgG Fc-binding proteins were encoded by fcrA and emmL, the product of enn was not identified. The presence of three emm-related genes in this region is reminescent of vir regulon organization in OF⁺ rather than OF^? strains as earlier defined by others. Furthermore, analysis of the deduced product of the emmL gene showed deletions and amino acid substitutions within the PGTS-rich domain and membrane anchor, which thus resembles corresponding products of OF⁺ rather than OF^? strains. In view of these findings, the opacity factor (OF) activity of the strain was tested using growth supernatant, with negative outcome. However, a concentrated SDS cell extract revealed definite OF activity. One of two other type M15 reference strains also showed definite OF activity in SDS extracts. We therefore propose that type M15 strains belong to the OF⁺ category but often show low levels of expression of OF.     

Schistosoma mansoni: identification of immunoglobulins associated with the tegument of adult parasites from mice.

Immunocytochemical and immunodiffusion studies were conducted in an attempt to identify the immunoglobulins associated with the tegumental surfaces of Schistosoma mansoni. Peroxidase-labeled monospecific rabbit anti-mouse immunoglobulin class or subclass sera revealed the presence of mouse IgG₁, IgG₂, IgG₃, IgA, and IgM on the surface of adult parasites recovered from mice. These observations were confirmed by double gel diffusion of the various rabbit antisera against an eluate obtained from mouse worms.     

Guinea pig peritoneal macrophages. Differential effects of lectins on interaction with IgG immunoglobulins

Guinea pig peritoneal macrophages have on their surface two receptors, one (Fcγ₁/γ₂R) binding both guinea pig IgG1 and IgG2 and the second (Fcγ₂R) binding only IgG2 immunoglobulins. We have previously shown that treatment of macrophages with neuraminidase or glycosylation inhibitors affects, in a different way, the binding of guinea pig IgG1, IgG2, and rabbit IgG. In the present study we have shown that pretreatment of guinea pig macrophages with lectins (Con A, WGA, and PNA) also has a different effect on the interaction of the cells with IgG. The lectins increased the binding of guinea pig IgG1, whereas rabbit IgG and guinea pig IgG2 were bound with a lower efficiency than in the case of control cells. Since sialic acid residues seem to modulate the activity of receptors and WGA interacts with sialylated oligosaccharides, we determined the IgG-binding characteristics for WGA-pretreated macrophages. We found that the increase in IgG1-binding ability was caused by an increase in the value of K_app, but the number of IgG-binding sites was lower than in the control cells. In the case of rabbit IgG and guinea pig IgG2 we observed a decrease of both the value of K_app and the number of IgG-binding sites. WGA did not interact directly with the Fcγ receptor. The results of our former papers and the different effects of lectins of various specificities described in this paper suggest different positions of Fcγ₁/γ₂ and Fcγ₂R in the plane of the plane of the macrophage membrane in respect to various membrane glycoconjugates. Interaction of IgG with macrophage Fcγ receptors depends in a different way on glycoconjugates on the surface of the macrophage. Our results suggest that changes in glycosylation of macrophage surface glycoconjugates may be used by the cell for regulating the binding activities of the macrophage Fcγ receptors.     

Relationships between IgE/IgG4 Epitopes,Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family

Background

Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies.The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown.The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG₄ binding regions.

Methodology/Principal Findings

The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg²⁺, but not Ca²⁺, binding was determined by band shift using SDS-PAGE. IgE and IgG₄ epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients.The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG₄. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera.

Conclusions/Significance

This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG₄ binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.     

IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses,Fc-Glycans and a Fab-Peptide Sequence

The Fc-glycan profile of IgG₁ anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG₁, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG₄ peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG₄, the ACPA-IgG₄ Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG₁ compared to FT-IgG₁ were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG₁ relative to FT-IgG₁ as well as lower content of IgG₂ (p = 0.0000001) and elevated content of IgG₄ (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG₁ and ACPA-IgG₄ are distinct. Given that IgG₁ and IgG₄ have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.