CNBr cleavage of the light chain of human complement factor I and alignment of the fragments.

The primary structure of the second component of human complement (C2) was determined by cDNA cloning and sequence analysis. C2 has 39% identity with the functionally analogous protein Factor B. The C-terminal half of C2a is homologous to the catalytic domains of other serine proteinases. C2b contains three direct repeats of approx. 60 amino acid residues. They are homologous to repeats in Factor B, C4b-binding protein and Factor H, suggesting a functional significance of the repeat in C4b and C3b binding. The repeats are also found in the non-complement proteins beta 2-glycoprotein I and interleukin-2 receptor, and this repeat family may be widespread.     

Regulation of human Ig lambda light chain gene expression by NF-kappa B.

The human Iglambda enhancer consists of three separated sequence elements that we identified previously by mapping DNase I-hypersensitive regions (HSS) downstream of the C region of the Iglambda L chain genes (HSS-1, HSS-2, and HSS-3). It has been shown by several laboratories that expression of the H chain genes as well as the kappa genes, but not the lambda genes, is dependent on constitutive NF-kappaB proteins present in the nucleus. In this study we show by band-shift experiments, in vivo footprinting, and transient transfection assays that all three hypersensitive sites of the human Iglambda enhancer contain functional NF-kappaB sites that act synergistically on expression. We further show that the chicken lambda enhancer also contains a functional NF-kappaB site but the mouse lambda enhancer contains a mutated, nonfunctional NF-kappaB site that is responsible for its low enhancer activity. It is possible that the inactivating mutation in the mouse Iglambda enhancer was compensated for by an expansion of the Igkappa L chain locus, followed by a contraction of the Iglambda locus in this species.     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.     

The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.     

Site-specific N-glycan characterization of human complement factor H

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.     

Allelic polymorphisms and RFLP in the human immunoglobulin lambda light chain locus

The organization of the human immunoglobulin lambda light chain locus (IGL) was recently described. This locus has been entirely sequenced. To evaluate the extent of the genomic variability existing inside that locus, we compiled all the available sequences of germline IGLV genes to find variants of Vλ sequences. We also looked for RFLP polymorphisms in a reputedly highly polymorphic human population from eastern Senegal, and compiled all RFLP data previously published. Analysis of these data indicates that IGLV alleles are frequent and increase the diversity of the lambda light chain repertoire in the human population. In contrast, RFLP and polymorphism by insertion and/or deletion are limited in that locus. This observation reinforces our hypothesis that the human IGL locus has undergone less evolutionary shuffling than the human kappa or heavy-chain loci. Received: 23 December 1998 / Accepted: 1 March 1999     

Molecular cloning of a human serum protein structurally related to complement factor H.

Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H.     

Intergenic exchange maintains identity between two human lambda light chain immunoglobulin gene intron sequences.

Evidence for gene conversion or unequal double crossover in the human lambda light chain immunoglobulin locus is presented. The high level of J2C2-J3C3 intron cross-hybridization, the identity of the J lambda and J lambda 3 coding and intron sequences, the presence of multiple base differences between the C lambda 2 and C lambda 3 coding regions, and the presence of both the unconverted and converted alleles in the normal gene pool, suggest that a recombinational event has resulted in the conversion of the J lambda 2 coding and intron sequences to those of J lambda 3 and its flanking sequences. Intergenic exchanges, such as the one described here, may provide a mechanism to maintain sequence homogeneity and functionality among the duplicated members of the human lambda gene family.     

Biases in Ig lambda light chain rearrangements in human intestinal plasma cells

Human intestinal lamina propria plasma cells are considered to be the progeny of chronically stimulated germinal centers located in organized gut-associated lymphoid tissues such as Peyer's patches and isolated lymphoid follicles. We have sampled human colonic lamina propria plasma cells and naive and memory B cell subsets from human Peyer's patches by microdissection of immunohistochemically stained tissue sections and used PCR methods and sequence analysis to compare IgVlambdaJlambda rearrangements in the plasma cell and B cell populations. Rearrangements that were either in-frame or out-of-frame between V and J were compared. Usage of IgVlambda families in the in-frame rearrangements from the plasma cells resembled that observed in the mantle cells, suggesting that antigenic selection for cellular specificity does not dramatically favor any particular Vlambda segment. However, in marked contrast, out-of-frame rearrangements involving Vlambda1 and Vlambda2 families are rarely observed in intestinal plasma cells, whereas rearrangements involving Vlambda5 are increased. This resulted in significantly biased ratios of in-frame:out-of-frame rearrangements in these Vlambda families. Out-of-frame rearrangements of IgVlambdaJlambda from plasma cells, including those involving the Vlambda5 family, have a significant tendency not to involve Jlambda1, consistent with the hypothesis that this population includes rearrangements generated by secondary recombination events. We propose that modification of out-of-frame rearrangements of IgVlambdaJlambda exists, probably a consequence of secondary rearrangements. This may be a mechanism to avoid translocations to susceptible out-of-frame IgVlambdaJlambda rearrangements during somatic hypermutation.     

Serologic and chemical differentiation of human lambda III light chain variable regions

Human lambda L chains of a major V lambda subgroup, V lambda III, have been differentiated serologically and chemically into three V lambda III sub-subgroups designated V lambda IIIa, V lambda IIIb, and V lambda IIIc. Antisera prepared against lambda III Bence Jones proteins were obtained that recognized distinctive V lambda III-related epitopes expressed by monoclonal lambda III L chains. After appropriate absorption, these reagents were rendered specific for three distinct populations of lambda III proteins--lambda IIIa, lambda IIIb, and lambda IIIc. The antisera were used in comparative immunodiffusion analyses of 28 monoclonal lambda III L chains, 10 of which were classified as lambda IIIa, 4 as lambda IIIb, and 14 as lambda IIIc. The isotypic nature of the three lambda III sub-subgroups was demonstrated serologically through analyses of lambda-chains derived from the serum IgG molecules of normal individuals. The amino acid sequences of five serologically classified lambda III chains, which included members of the three V lambda III sub-subgroups, had been previously determined. This information, in addition to our establishment of the complete (or virtually complete) V region sequence of 15 and the partial sequence of eight other lambda IIIa, lambda IIIb, and lambda IIIc proteins, made it possible to correlate chemical data with serologic classification. Proteins within each of the three serologically-classified lambda III sub-subgroups typically possessed a high degree (approximately 83%) of intra-sub-subgroup sequence homology that included both framework and complementarity determining region residues. Furthermore, within the framework and complementarity determining regions, sub-subgroup-specific residues were identified. Taken together, these data reveal that the human V lambda III genome consists of (at least) three distinct V lambda IIIa, V lambda IIIb, and V lambda IIIc germline genes that encode for lambda IIIa, lambda IIIb, and lambda IIIc L chains, respectively.     

Chromosomal orientation of the lambda light chain locus: V lambda is proximal to C lambda in 22q11.

We have demonstrated that the chromosomal breakpoint at 22q11 of a Burkitt lymphoma cell line (PA682) with an 8;22 translocation interrupts the variable region of the lambda light chain locus. In these cells, all of the C lambda and some V lambda sequences translocate to the 8q+ chromosome whereas some V lambda sequences remain on the 22q-. These results indicate that the lambda light chain locus on the long arm of chromosome 22 is oriented such that V lambda is proximal to C lambda.     

Primary structure of a human IgA1 immunoglobulin. V. Amino acid sequence of a human IgA lambda light chain (Bur).

The sequence of the lambda light chain of the Bur IgA1 molecule has been determined. It comprises 214 amino acid residues with a blocked NH2 terminus and lacks carbohydrate. The V-region sequence is of the VlambdaII subgroup and contains the coupled interchanges Arg-7 and Cys-87. The Lv3 region is comparatively short and hydrophobic in nature and lends support for the designation of this area as a hypervariable deletion region. The C-region exhibits the Mcg+ Kren+ Oz- isotypes. These appear coupled with substitution at position 100 (in the V-region). The pattern of nonrandom association of V- and C-regions and H and L chains is discussed in terms of the generation of antibody diversity. With the companion papers in this series, the complete primary structure of a human IgA1 molecule is established.     

Primary structure of the variable region of a human lambda VI light chain: Bence Jones protein SUT

To ascertain if lambda VI light chains have unique structural features that account for the preferential association of these proteins with primary or multiple myeloma-related amyloidosis (amyloidosis AL) we have determined the complete amino acid sequence of the variable (V) region of the lambda VI Bence Jones protein SUT. This protein, obtained from a patient with amyloidosis AL, represents a complete light chain consisting of 216 residues and it has structural and serologic properties characteristic for lambda VI light chains. The sequence of the joining segment (J) (positions 100 to 111) of protein SUT is identical to that of the J lambda I segment of the mouse IG lambda light chain gene. V region SUT is closely homologous in sequence to that of another lambda VI amyloid fibrillar protein, AR, differing by 21 residues. The V regions of proteins SUT and AR contain a two-residue insertion at positions 68 and 69 that has also been found in two other lambda VI human light chains but not in the lambda-chains of other V region subgroups.     

Mcg light chain dimer as a model system for ligand design: a docking study

Mcg light chain dimer has been extensively studied by crystallography and peptide binding studies to investigate its peptide cross-reactivity as well as to use it as a model system for designing space filling peptide ligands. Here we extend these investigations by utilizing automated docking. Mcg light chain dimer is an ideal model system for such study due to the availability of experimental data for both the native structure and the 14 complexes with various peptide ligands. We show the ability of the docking approach to reproduce the experimental structures and discuss the limitations associated with such outcomes. We demonstrate the usefulness of the docking approach in generating structural information otherwise not available from the experiment.     

The unique region of surrogate light chain component lambda5 is a heavy chain-specific regulator of precursor B cell receptor signaling

Signals transduced by precursor-BCRs (pre-BCRs) composed of Ig mu heavy chains (HCs) and the surrogate L chain components lambda5 and VpreB are critical for B cell development. A conserved unique region (UR) of lambda5 was shown to activate pre-BCR complexes in transformed cells and to engage putative ligands, but its contribution to pre-B cell development is not known. It is also not clear why the lambda-like sequences in lambda5 are used to select HCs that will associate mainly with kappa L chains. In this study, we show that, in transformed and primary mouse B cell progenitors, receptors containing full-length HCs and lacking the lambda5UR were expressed at higher surface levels, but exhibited reduced activity compared with normal pre-BCRs in supporting developmental changes that accompany the progenitor to pre-B cell transition in primary cell culture systems and in the bone marrow in vivo. In contrast, deletion of the lambda5UR did not change net signaling output by the Dmu-pre-BCR, a developmentally defective receptor that exhibited impaired activity in the primary cell culture system. Moreover, the lambda-like sequences in lambda5 were more accommodating than kappa in supporting surface expression and signaling by the different HCs. These results show that the lambda5UR is important, although not essential, for surrogate L chain-dependent receptor signaling in primary cells, and furthermore may help allow discrimination of signaling competency between normal and Dmu-pre-BCRs. That the lambda-like portion of lambda5 in the absence of the UR was nondiscriminatory suggests that the lambda5UR focuses pre-BCR-dependent selection on the HC V region.     

Species-specific interaction of Streptococcus pneumoniae with human complement factor H

Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway inhibitor. S. pneumoniae recruits FH through a previously defined FH binding domain of choline-binding protein A (CbpA), a major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH, but not to the FH proteins of mouse and other animal species tested to date. Accordingly, deleting the FH binding domain of CbpA in strain D39 did not result in obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal fluid. Finally, our phagocytosis experiments with human and mouse phagocytes and complement systems provide additional evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-mediated host defense in humans.     

Haemophilus influenzae interacts with the human complement inhibitor factor H

Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6-7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18-20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of approximately 32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.