Yoshihiro Fukuoka

Structural polymorphism of mouse complement C2 detected by microscale peptide mapping: Linkage to H-2

Complement C2 was isolated from 17 mouse strains by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined for structural polymorphism by using micro-peptide mapping. By comparing the peptide maps of tryptic digests of C2 from various strains, two allotypic variations were detected. B 10 and 14 other mouse strains demonstrated C2.1 type, while a wild mouse line (M.Mol-Ohm) and one BIO congenic strain, B10.MOL.OHM, which carries the H-2 derived from M.Mol-Ohm, demonstrated C2.2 type. (B10 × Bl0.MOL.OHM)F1 demonstrated codominantly expressed C2 type (C2.1.2). Desialation of mouse C2 did not abolish the observed variation of mouse C2. It is concluded that an H-2-linked codominant locus controls the structure of mouse complement C2, further confirming the extensive homology of the major histocompatibility complex among higher vertebrate species.

Active recombinant C3a of human anaphylatoxin produced in Escherichia coli

DNA sequence coding for the complete human C3a with 77 amino acids was divided into three portions, synthesized separately and constructed for expression in Escherichia coli. High expression of the recombinant C3a was achieved by an expression system using T7 polymerase. Purified recombinant C3a showed the same activities of ileum contraction and platelet aggregation of guinea pig as C3a purified from human serum.

Expression of biologically active C3a as fusion proteins.

We selected three kinds of plasmids for expression of C3a as fusion proteins. The proteins were purified by affinity chromatography using the respective specific resins, and their activities were measured by guinea pig platelet aggregation. We showed that polyhistidine (polyHis)-C3a fusion protein was able to exhibit 30% of the activity of natural C3a. However, glutathione S-transferase (GST)-C3a fusion protein exhibited only 10% of such activity, and no activity was measured for maltose binding protein (MBP)-C3a fusion protein. The purified polyHis-C3a fusion protein was attached to the Ni-NTA agarose column in an attempt to isolate the C3a receptor from guinea pig platelets. The C3a binding protein isolated from digitonin-solubilized guinea pig platelet membrane was approximately 50 kDa on SDS-polyacrylamide gel. This is the first report of C3a fusion protein production with biological activity.

Anaphylatoxin C3a induces rapid protein phosphorylation in guinea pig platelets.

We investigated C3a signal transduction using guinea pig platelets. Anaphylatoxin C3a induced cytosolic calcium influx and turnover of inositol phospholipids and caused protein phosphorylation via specific C3a receptors on guinea pig platelets. Phosphorylation of 40 kDa and 20 kDa proteins was detected within 30 s after C3a stimulation. From phosphoamino acid analysis of both proteins, it was found that about 80% was serine and 20% was threonine. Phosphorylation was decreased by pretreatment of platelets with calcium blockers, diltiazem and TMB-8. A protein kinase C inhibitor, staurosporine, effectively blocked phosphorylation of both proteins, but H-7 did not. The effects of these inhibitors on platelet aggregation were correlated with their effects on protein phosphorylation. These results suggest that protein-serine/threonine phosphorylation is important for C3a signal transduction in guinea pig platelets and that the isoenzyme of protein kinase C or another kinase probably participates in such protein phosphorylation. C3a also caused protein-tyrosine phosphorylation of 56 kDa and 33-36 kDa proteins within 30 s. Staurosporine effectively blocked phosphorylation of these bands. It is suggested that tyrosine kinase also may be involved in C3a signalling in guinea pig platelets.

Nucleotide sequence analysis of a human monoclonal antibody 22-13 reactive with lung tumor-associated antigen

A human monoclonal antibody (HuMAb) 22-13 (IgG1, kappa) recognizes a cytoplasmic antigen associated primarily with human lung tumors. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMAb 22-13. This HuMAb uses a VH gene member of the V(H)Ia gene family, 51P1 and is productively rearranged with a D-D fusion product of the D(LR)2 and D(XP)2 germ line DH genes and the germ line JH3 gene. HuMAb 22-13 Vkappa belongs to the kappa light chain variable subgroup IIIb family and appears to be derived from the Humkv325 germ line gene and is rearranged with a germ line Jkappa5 gene. The results reveal that production of a HuMAb 22-13 is achieved by rearrangement of the 51P1/Humkv325 germ line variable region gene combination, associated with the autoimmune repertoire and that HuMAb 22-13 has a striking sequence homology to rheumatoid factors (RFs) of the Wa idiotypic family. HuMAb 22-13 and Wa RFs have in common V(H)Ia and VkappaIIIb gene segments, but use different DH, JH and Jkappa gene segments. However, in spite of this structural similarity, HuMAb 22-13 does not display rheumatoid factor activity. Taken together with the reported findings, these data indicate the representation of the shared usage of highly homologous variable region genes in entirely different humoral immune responses in the human system.

Activation of guinea pig platelets by a monoclonal antibody

We herein describe the characterization of a monoclonal anti-guinea pig platelet antibody, termed GT2-12, which causes aggregation, ATP and [3H]serotonin release, and platelet protein phosphorylation. GT2-12 can bind to platelets and megakaryocytes in guinea pig bone marrow cells. A protein of 34,000 daltons was detected by immunoprecipitation of platelet lysates with GT2-12. Incubation of 32P-labeled guinea pig platelets with GT2-12 resulted in rapid phosphorylation of proteins of 40,000 and 20,000 daltons. GT2-12-induced aggregation and protein phosphorylation of platelets were inhibited by diltiazem, TMB-8 and dibutyryl cAMP and partially inhibited by indomethacin. The F(ab)2 fragment of GT2-12 IgG also induced platelet aggregation, indicating that activation is not mediated by Fc receptors. This type of antibody should be useful for studying platelet function in the guinea pig model.

Nucleotide sequence analysis of a human monoclonal antibody TONO-1 with cytotoxic potential for T-leukemia/lymphoma cells.

A human monoclonal antibody (HuMab) TONO-1 (IgM, lambda) recognizes cell surface antigens associated primarily with human T-leukemia/lymphoma cells. In this study, we investigated the reactivity against T-leukemia/lymphoma cells in detail, cytotoxic potential and primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMab TONO-1. Expression of the molecules (TONO-1 Ags) detected by a HuMab TONO-1 was significantly heterogeneous even in the same T-leukemia/lymphoma cell lines HPB-MLT and MOLT-4F. The flow cytometric curves showed an unusual broad-based spread of fluorescence intensity. HuMab TONO-1 was shown to have the ability to kill the T-leukernia/lymphoma cells efficiently in the presence of rabbit complements. However, HuMab TONO-1 did not demonstrate significant antibody-dependent cellular cytotoxic activity. Furthermore, HuMab TONO-1 heavy and light chain variable regions were cloned, sequenced and analyzed. HuMab TONO-1 uses a V(H) gene member of the V(H)IV gene family V(H)71-4, and is productively rearranged with the germ line D(H) gene D(XP)1, and the germ line J(H)5 gene with multiple somatic mutations. HuMab TONO-1 Vlambda belongs to the lambda light chain variable subgroup I family and is derived from the Vlambdalc germ line gene Humlv1042, and germ line gene Jlambda1 without somatic mutations. The results reveal that the production of HuMab TONO-1, with cytotoxic potential for human T-leukemia/lymphoma cells, is achieved by rearrangement of the V(H)71-4/Humlv1042 germ line variable region gene combination, that is associated with the autoimmune repertoire.

Presence of C5a‐potentiating activity in the plasma of a patient with Kimura's disease

To determine whether the response to the active complement fragment C5a in polymorphonuclear leukocytes (PMN) obtained from a patient with Kimuras disease is similar to that in normal subjects, we evaluated superoxide anion (O2‐) generation after stimulation with C5a. The patients PMN produced more O2‐ than did those from healthy controls after stimulation with human C5a in vitro. The response returned to normal with the improvement in clinical indicators after initiation of steroid administration. We conducted the following experiments with the plasma of this patient during the active disease stage to establish the presence of a humoral factor that potentiated the C5a‐response of PMN. Incubation of PMN from normal controls having the same blood type as the patient at 37°C for 2 h with the patients plasma at the active disease stage revealed that the cells generated abundant O2‐ after stimulation with C5a. Incubation of normal PMN with the patients plasma during the inactive disease stage showed no potentiated response to C5a. This activity was lost after incubation at 56°C for 30 min. Coincubation of PMN with methylprednisolone up to 100 μg/ml did not suppress this activity. Although the plasma concentration of C5a at the active stage was mildly elevated (13 ng/ml), it was below the limit of detection (<10 ng/ml) at the inactive stage. These results suggest the presence of a heat‐labile, humoral factor in the patients plasma that upregulated the response of PMN to C5a and that was not suppressed by in vitro treatment with a steroid. This factor may influence the acute inflammatory reaction in this disorder.