Pak H. Poon

Identification of defensin binding to C1 complement

In human serum we found strong defensin binding to the complexes of activated C1 complement (C ) and C1 inhibitor (C1i). Purified C1q, activated C1 tetramer ( 2 2) and C1i did not bind defensin. When ( 2 2) was dissociated by EDTA, only the activated C1s (C s) bound defensin. Binding of defensins to C complement represents a newly recognized bridge between the complement‐ and phagocyte‐mediated host defenses, and a potential mechanism for protecting infected tissue from cytotoxic injury by defensin.

Semi-flexible joint in the C1q subunit of the first component of human complement

Abstract C1q appears in electron micrographs in two different projections: lateral projections, in which the molecules resemble bunches of tulips; and top projections, in which the molecules are seen as six terminal subunits connected to a central portion. We have measured 39 particularly well-formed top views to determine the distribution of distances of terminal subunits from the central portion, from which may be calculated the distribution of the angles made by the connecting strands with an axis through the central portion. This distribution peaks sharply at a preferred angle of 50 °. A limited degree of flexibility must exist, however, for a few molecules are found with angles ranging from 20 ° to 80 °. Therefore, we suggest the existence of a semi-flexible joint at the point of interruption of the collagen-like amino acid sequence where the connecting strands join to the central portion of the C1q molecule.

Conformation and restricted segmental flexibility of C1, the first component of human complement

Seventy selected images of chemically crosslinked C1 are analyzed to illustrate structural details of the C1qC1r2C1s2 complex. From inspection of these images, the C1r2C1s2 tetramer can be seen to be located in the region of the C1q arms, cleanly separated from the C1q heads and from at least 90%, if not all, of the C1q stem. From measurements made upon 65 images, the semicone angles formed between the spreading arms and the symmetry axis passing through the stem of C1 may be calculated. Unlike C1q, for which a wide variety of angles is found, the C1 complex appears to possess a restricted range of angular flexibility with an average value of about 50 degrees. The volume inside the cone formed by the spreading arms of C1q is too small to contain the entire C1r2C1s2 tetramer; at least some of the tetramer must lie outside the cone when it is bound to C1q to form C1. From our knowledge of the sizes and structures of its subunits, and from symmetry considerations, a model is proposed for the configuration of the C1 complex in which the middle portion of the C1r2C1s2 tetramer is centrally located among the arms close to the stem of the C1q and with the two protruding ends of the tetramer wrapped around the outside of the cone. Functional implications of this more rigid structure are discussed with relevance to C1q-induced aggregation of latex beads and C1-induced disaggregation.

Domain Identification of Hormone-sensitive Lipase by Circular Dichroism and Fluorescence Spectroscopy, Limited Proteolysis, and Mass Spectrometry

Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Østerlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411–420; Contreras, J. A., Karlsson, M., Østerlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426–31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1–4 and 5–9, respectively, of which the latter contains an exposed regulatory module outside the catalytic α/β-hydrolase fold core.

Expression and characterization of a 159 amino acid, N-terminal fragment of human complement component Cls☆

Abstract A 159 residue, N-terminal fragment of the human Cls complement component. Clsα(159), was expressed in the baculovirus, insect cell system. The protein was abundantly produced 3 days after infection, reaching levels as high as 40 μg/ml in cell culture media. It had a molecular weight of 18.100 (±4.9) Da by laser desorption mass spectrometry, close to the theoretical value of 18.111 Da, confirmed by sequencing. Sedimentation equilibrium and gel filtration column chromatography showed that Clsα(159) was a monomer in the presence of EDTA, and a dimer in the presence of Ca2+. The Clsα(159)2 dimer had a sedimentation coefficient of 3.1 S. When the Clsα(159)2 was mixed with Clq, there was little or no interaction. Likewise, unactivated Clr2 dimer had a sedimentation coefficient of 6.8 S, and when mixed with Clq little or no interaction was observed. When Clsα(159)2 was mixed with the 6.8 S Clr2 in Ca2+, a 7.5 S complex was formed, presumably the Clsα(159)·Clr·Clr·Clsα(159) tetramer. When Clq, which migrated at 10.1 S was mixed with Clsα(159)2 and Clr2 in the presence of Ca2+, a Cl-like complex, but containing Clsα(159) instead of Cls, was formed which migrated at 14.0 S. This Cl-like molecule remained unactivated unless challenged with an ovalbumin-antiovalbumin immune complex. In the presence of immune complex, the Clr became activated. This suggested that the presence of the 159 amino acid Clsα domain, which held the Clr to the Clq, was sufficient to permit activation by an immune complex, even though the catalytic domains of Cls were not present.

Isolation of human complement subcomponents C1r and C1s in their unactivated, proenzyme forms

We have modified a standard isolation procedure for C1r and C1s, which employs IgG-Sepharose affinity chromatography followed by DEAE chromatography. As usual, all steps were performed at low temperature and two proteolytic inhibitors, PMSF and NPGB, were added during affinity chromatography on IgG-Sepharose. The novel condition was to keep the pH at pH 6.1 during the entire procedure, where activation was markedly depressed. In addition, purification was improved by washing the IgG-Sepharose column with a buffer free of added divalent cations immediately prior to elution of the C1r and C1s with EDTA. The final yields of highly purified C1r and C1s were about 20%; little or no activated material was detected in these highly purified fractions.