Characterization of Binding Properties of Individual Functional Sites of Human Complement Factor H.

Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is flexible enough that it folds back on itself. Factor H regulates complement activation through its interactions with C3b and polyanions. Three binding sites for C3b and multiple polyanion binding sites have been identified on Factor H. In intact Factor H these sites appear to act synergistically making their individual contributions difficult to distinguish. Recombinantly expressed fragments of human Factor H were examined using surface plasmon resonance (SPR) for interactions with C3, C3b, iC3b, C3c, and C3d. Eleven recombinant proteins of lengths from one to twenty domains were used to show that the three C3b-binding sites exhibit 100-fold different affinities for C3b. The N-terminal site [complement control protein (CCP) domains 1-6] bound C3b with a Kd of 0.08 µM and this interaction was not influenced by the presence or absence of domains 7 and 8. Full length Factor H similarly exhibited a Kd for C3b of 0.1 µM. Unexpectedly, the N-terminal site (CCP 1-6) bound native C3 with a Kd of 0.4 µM. The C-terminal domains (CCP 19-20) exhibited a Kd of 1.7 µM for C3b. We localized a weak third C3b binding site in the CCP 13-15 region with a Kd estimated to be ~15 µM. The C-terminal site (CCP 19-20) bound C3b, iC3b, and C3d equally well with a Kd of 1 to 2 µM. In order to identify and compare regions of Factor H that interact with polyanions a family of 18 overlapping three domain recombinant proteins spanning the entire length of Factor H were expressed and purified. Immobilized heparin was used as a model polyanion and SPR confirmed the presence of heparin binding sites in CCP 6-8 (Kd 1.2 µM) and in CCP 19-20 (4.9 µM) and suggested the existence of a weak third polyanion binding site in the center of Factor H (CCP 11-13). Our results unveil the relative contributions of different regions of Factor H to its regulation of complement, and may contribute to the understanding of how defects in certain Factor H domains lead to disease.

Quadriparesis in a young man due to chronic inflammatory demyelinating polyneuropathy.

A man of 30 years, admitted in Neurology unit of Mymensingh Medical College Hospital with weakness of all four limbs for 4 weeks which was of gradual onset and progressive. He had no difficulty in vision, swallowing & speech. He had no disturbances of sensation, bowel & bladder functions. There was no preceding history of gastrointestinal or upper respiratory tract infection or vaccination. General examination was normal except the presence of hypertension detected two months before the onset of current illness. All cranial nerve functions were intact. Muscle power was grade 4 in all limbs and the reflexes were absent. All modalities of sensation and coordination were normal. Cerebro-spinal fluid (CSF) study revealed protein-cell dissociation. Electro physiologic findings were consistent with Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). The patient was treated with prednisolone 60mg/day for two months with improvement of muscle power. The steroid was reduced gradually and then maintained 20mg/day without any relapse.

Polyanion-induced self-association of complement factor H.

Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin, and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This result was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18-20) also exhibited polyanion-induced self-association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H.

A novel vector for the expression of SCR domains in insect cells.

Exploitation of recombinant technology to study proteins containing strings of short consensus repeat (SCR) domains largely depends on expression vectors. In this paper, we describe a vector for cloning and constitutive expression of single or multiple SCR domains. The recombinant vector has unique additive features over commercially available vectors that make it a universal cloning vector for SCR domains as well as a vector suitable for expressing any protein fragment beginning and ending with cysteine residues. As a demonstration of its usefulness, the constitutive extracellular expression of five SCR-containing proteins derived from complement factor H is presented.

Lung surfactant protein B promoter function is dependent on the helical phasing, orientation and combinatorial actions of cis-DNA elements.

Surfactant protein B (SP-B), a hydrophobic protein of lung surfactant, is essential for surfactant function, normal respiration and survival. SP-B is expressed in a cell-type specific manner by the alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. Our previous studies showed that the activity of the rabbit SP-B minimal promoter (-236/+39 bp) is dependent on the binding of an array of transcription factors including Sp1, Sp3, thyroid transcription factor 1, hepatocyte nuclear factor 3 and activating transcription factor/cyclic AMP response element binding protein. The SP-B minimal promoter sequence as well as the spacing and orientations of cis-DNA elements are conserved in human, rabbit and mouse SP-B genes. In the present study, we investigated the importance of spacing and orientation of cis-DNA elements on SP-B promoter function in NCI-H441 cells, a human cell line of Clara cell lineage. Further we investigated the effects of transcription factors on SP-B promoter expression by co-transfection experiments. Results showed that disruptions of helical phasing and orientation of cis-DNA elements reduced SP-B promoter activity indicating that proper alignment and orientation of cis-DNA elements are necessary for SP-B promoter function. Co-transfection experiments showed that transcription factors function in a combinatorial rather than in a synergistic manner to enhance SP-B promoter activity.