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O H Brekke

Publications and source records attributed to O H Brekke.

11 recordsLinked to original sources
PubMed2000

Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation.

The classical complement activation cascade of the immune system is initiated by multivalent binding of its first component, C1q, to the Fc region of immunoglobulins in immune complexes. The C1q binding site on mouse IgG2b has been shown to contain the amino acids Glu 318, Lys 320 and Lys 322 in the C(H)2 domain (Duncan, A.R., Winter, G.,1988. The binding site for C1q on IgG. Nature 322 738-740). Identical or closely related motifs are found on all IgGs in all species, and the binding site has therefore been thought to be universal. However, the results from another study indicate that the site is different in human IgG1 molecules (Morgan, A., Jones, N.D., Nesbitt, A.M., et al., 1995. The N-terminal end of the C(H)2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding. Immunology 86 319-324). To determine the site(s) responsible for complement activation in anti-NIP-mouse/human IgG3 antibodies, we have mutated amino acids Lys 276, Tyr 278, Asp 280, Glu 318, Lys 320 and Lys 322 in two beta-strands in the C(H)2 domains of human IgG3. In addition, we mutated the Glu 333, which resides in close proximity to the postulated C1q-binding site of mouse IgG2b, as well as Leu 235 in the lower hinge region. All mutants were tested in Antibody Dependent Complement Mediated Lysis (ADCML)(4) assays, where the antigen concentration on target cells was varied and human serum was complement source. Only the mutants that lacked the positively charged side chain of lysine in position 322 showed strong reduction in ADCML, particularly at low antigen density on target cells. Alanine scanning of positions 318 and 320 did not affect ADCML, contrary to what was observed for mouse IgG2b. Neither did a leucine to glutamic acid mutation in position 235 have the effect that has been reported for human IgG1. These results suggest that the complement binding site on human IgG3 molecules is different from that found on mouse IgG2b, and possibly on human IgG1 as well. Thus the contact site may not be conserved.

Binding Sites↗
PubMed1998

Comparisons of the ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form small immune complexes: a role for flexibility and geometry.

Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb, and the resultant immune complexes were analyzed by negative stain immunoelectron microscopy. Complexes were scored for their geometry (linear versus ring complexes) and size (dimer, trimer, etc.). Ring dimers are the thermodynamically most favorable configuration, unless inhibited by steric and/or flexibility constraints. We found ring dimerization to correlate with the length of the upper, but not middle or lower, hinge. In contrast, the geometry and size of complexes of those molecules lacking formal hinges were unpredictable. A hingeless IgG mutant and native IgE readily formed ring dimers. Remarkably, monomeric IgM formed more ring dimers than any of the other Igs tested, including IgG3. We also tagged the Fab arms and measured the mean Fab-Fab angles and the degree of angular variation for each type of Ig. Surprisingly, IgM proved the most flexible by this assay. In hinged Igs, there was a correlation between length of the upper hinge and Fab-Fab flexibility. In contrast, we found no correlation between the mean Fab-Fab angle in uncomplexed Igs and their ability to dimerize with anti-Id mAb. These data suggest that the physicochemical methods typically used to evaluate molecular flexibility are often of low predictive value when tested in a functional assay.

Amino Acid Sequence↗
PubMed1997

Identification and characterisation of C1q-binding phage displayed peptides.

Five phage displayed peptide libraries were screened for binders to C1q, the recognition subunit of the classical complement pathway. Two rounds of panning resulted in the isolation and characterisation of several different phage displayed C1q-binding peptides from all five libraries. Two groups of the characterised peptides show sequence similarity with part of the metal ion dependent adhesion site (MIDAS) of integrin A-domains, and the site 187LRNPCPNKEKECQPPF of CD18 (integrin beta2), respectively. These results support binding of complement receptor 3 (CR3, CD11b/CD18, Mac1) to C1q and further suggest C1q binding sites in CR3. We also discuss sequence matches between the characterised peptides and proteins known to interact with C1q, as well as other proteins listed in the SwissProt databank. These findings are of interest for the study of the complement system and may lead to the development of peptides, fusion products or peptido-mimetics with C1q modulating potential.

Amino Acid Sequence↗
PubMed1995

The structural requirements for complement activation by IgG: does it hinge on the hinge?

The flexibility of antibody molecules principally derives from the structure of the hinge region. It has generally been accepted that the flexibility of the IgG hinge is necessary for complement activation to occur; however, recent studies dispute this premise. As described here by Ole Henrik Brekke, Terje Michaelsen and Inger Sandlie, it now appears that the only requirement of the hinge region for complement activation is the presence of inter-heavy-chain disulfide bond(s). Furthermore, the structural basis for the differences between IgG subclasses with respect to effector functions appear to be located within the CH2 domain of the immunoglobulin molecule.

Amino Acid Sequence↗
PubMed1994

One disulfide bond in front of the second heavy chain constant region is necessary and sufficient for effector functions of human IgG3 without a genetic hinge.

We have created four IgG3 mutants without a normal hinge region: (i) m0 without a genetic hinge; (ii) m0/C131S, where Cys-131 in m0 was mutated to Ser; (iii) m0/231C232 (formerly HM-1), where a Cys residue was inserted in m0 between Ala-231 and Pro-232; (iv) m0/C131S/231C232, which is a hybrid of m0/231C232 and m0/C131S. The wild-type IgG3 and all mutants bind 5-iodo-4-hydroxy-3-nitrophenacetyl groups. The wild type and mutants, m15 (with 15 aa in the hinge), m0/231C232, and m0/C131S/231C232, were all positive for complement-mediated lysis, antibody-dependent cellular cytotoxicity mediated by peripheral blood leukocytes, and phagocytosis by U937. m0/C131S/231C232 was only weakly positive and sometimes negative for respiratory burst activity mediated by peripheral blood neutrophils (polymorphonuclear leukocytes), whereas m15, m0/231C232, and wild-type IgG3 were strongly positive. The m0 and m0/C131S mutants were mainly negative for complement-mediated lysis, antibody-dependent cell-mediated cytotoxicity, and phagocytosis by U937 and polymorphonuclear leukocytes. The results indicate that a hinge spacer region is not necessary, but the correct alignment of the two second heavy chain constant regions in the IgG3 molecule by a minimum of one disulfide bond is necessary and sufficient for effector functions.

Amino Acid Sequence↗
PubMed1994

Human IgG isotype-specific amino acid residues affecting complement-mediated cell lysis and phagocytosis.

In this report we describe the construction of anti-5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) mouse/human immunoglobulin (Ig) G4 chimeric molecules with altered amino acid residues in the CH2 domain. Three mutants are described. Gln-268 is substituted by His in gamma 4 Q268H, Ser-331 is substituted by Pro in gamma 4 S331P, and in gamma 4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement-mediated cell lysis (CML) and phagocytosis by Fc gamma RII- and Fc gamma RIII-bearing polymorphonuclear leukocytes (PMN) were measured. In CML, gamma 4 Q268H was inactive, but both gamma 4 S331P and gamma 4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants gamma 4 S331P and gamma 4 Q268H/S331P were both active only when complement was introduced. gamma 4 Q268H was not active in phagocytosis under any conditions. We conclude that His-268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by Fc gamma RII and/or Fc gamma RIII. Pro-331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect Fc gamma RII/Fc gamma RIII-mediated phagocytosis. In this manner the mutants gamma 4 S331P and gamma 4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.

Animals↗
PubMed1993

Activation of complement by an IgG molecule without a genetic hinge.

The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

Animals↗
PubMed1993

The extended hinge region of IgG3 is not required for high phagocytic capacity mediated by Fc gamma receptors, but the heavy chains must be disulfide bonded.

Fc gamma receptor (Fc gamma R) phagocytosis and respiratory burst were induced by chimeric mouse-human anti-(4-hydroxy-5-iodo-3-nitrophenyl) acetyl IgG3 antibodies with mutations in hinge and/or in CH1 region. IgG3 mutants with different hinge length ranging from 47 to 0 amino acids, an IgG3 molecule with an artificial hinge of just one cysteine residue (HM-1), and two hybrid IgG3 molecules with IgG4 hinge or IgG4 CH1-hinge were tested. Using the monocytic cell line U937 as effector cells, the mutated IgG3 molecules were very similar, revealing high activity, while the IgG3/IgG4 hybrids revealed a slightly reduced activity. However, the hingeless (0-h) mutant was negative, except after interferon-gamma stimulation when it became slightly positive. Interestingly, HM-1 was as active as the IgG3 mutants. With polymorphonuclear leucocytes (PMN) as effector cells we obtained some day-to-day variations, but all the IgG3 mutants were highly active, with the two shortest hinge mutants somewhat less active. The IgG3/IgG4 hybrid molecules revealed an intermediate activity, while IgG4 wild-type and the 0-h mutant were negative. However, the HM-1 molecule revealed an activity similar to that of the IgG3 mutants. The phagocytic activity of U937 was inhibited by monomeric IgG, indicating the importance of Fc gamma RI. In contrast, with PMN both blockage of Fc gamma RII and cleavage of Fc gamma RIII were required to significantly reduce the phagocytosis and respiratory burst, thus showing that both receptors contribute to the effect. These results demonstrate that the extended IgG3 hinge region is not necessary for a high phagocytic activity and that the major structural importance of the hinge is to connect the two heavy chains in this region.

Disulfides↗
PubMed1993

Human IgG3 can adopt the disulfide bond pattern characteristic for IgG1 without resembling it in complement mediated cell lysis.

In this paper we describe the construction of mouse-human IgG3 mutant antibodies resembling IgG1 in their disulfide bond pattern between the heavy and light chain (H-L) and between the two heavy chains (H-H). The effector functions of these mutant antibodies were compared to normal IgG3 and IgG1. Changing only the disulfide bond pattern between the heavy and light chains did not alter the ability to induce complement mediated cell lysis (CML), regardless of the amount of corresponding antigen that had been introduced to the surface of the target cells. However, alteration of the disulfide bond pattern between the two heavy chains had a large effect on CML due to shortening of the hinge from 62 to 15 amino acids. No difference between the mutants and normal antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC) was observed. This suggests that IgG3 can adopt the H-L disulfide bond pattern of IgG1 without obtaining the CML activity characteristic for IgG1.

Amino Acid Sequence↗
PubMed1991

Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge.

We have altered the amino acid sequence of the hinge and the first constant domain (CH1) of mouse/human chimeric IgG3 antibodies by site-directed mutagenesis, so as to make the sequences identical to those of IgG4. All the mutant antibodies with altered hinge region were more active in complement activation and complement-mediated lysis than native IgG3. The mutations in CH1, however, did not alter the activity. This demonstrates the importance of the hinge region in modulating this effector function. The results show that the primary structure of neither CH1 nor the hinge of IgG4 is responsible for the lack of complement activation shown by this subclass.

Amino Acid Sequence↗