Elevated expression of Paneth cell CRS4C in ileitis-prone SAMP1/YitFc mice: regional distribution, subcellular localization, and mechanism of action.

Paneth cells at the base of small intestinal crypts of Lieberkühn secrete host defense peptides and proteins, including alpha-defensins, as mediators of innate immunity. Mouse Paneth cells also express alpha-defensin-related Defcr-rs genes that code for cysteine-rich sequence 4C (CRS4C) peptides that have a unique CPX triplet repeat motif. In ileitis-prone SAMP1/YitFc mice, Paneth cell levels of CRS4C mRNAs and peptides are induced more than a 1000-fold relative to non-prone strains as early as 4 weeks of age, with the mRNA and peptide levels highest in distal ileum and below detection in duodenum.

Bence Jones KWR protein structures determined by X-ray crystallography.

A Bence Jones protein isolated in the early 1960s from a patient (initials KWR) suffering from plasma-cell dyscrasia was crystallized and its structure was analyzed in four different unit cells by X-ray diffraction. The final models of the molecule in all crystal forms were virtually the same, although the elbow angles relating the constant and variable domains of the Bence Jones dimers varied over a range of 10 degrees. The tetragonal form had an R factor of 22.

Four crystal forms of a Bence-Jones protein.

Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4(3)2(1)2 and P2(1)2(1)2(1), with unit-cell parameters a = b = 68.7, c = 182.

Functional analysis of the alpha-defensin disulfide array in mouse cryptdin-4.

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function.

Paneth cell alpha-defensins from rhesus macaque small intestine.

Antimicrobial peptides are secreted by small intestinal Paneth cells as components of innate immunity. To investigate the role of alpha-defensins in enteric host defenses in nonhuman primates, alpha-defensin cDNAs were isolated, alpha-defensin peptides were purified from rhesus macaque small bowel, and the bactericidal activities of the peptides were measured. Six rhesus enteric alpha-defensin (RED) cDNAs, RED-1 to RED-6, were identified in a jejunum cDNA library; the deduced RED peptides exhibited extensive diversity relative to the primary structures of rhesus myeloid alpha-defensins.

Structural determinants of procryptdin recognition and cleavage by matrix metalloproteinase-7.

The bactericidal activity of mouse Paneth cell alphadefensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette, A.

Lipopolysaccharide-induced methylation of HuR, an mRNA-stabilizing protein, by CARM1. Coactivator-associated arginine methyltransferase.

The RNA-binding protein HuR stabilizes labile mRNAs carrying AU-rich instability elements. This mRNA stabilization can be induced by hypoxia, lipopolysaccharide, and UV light. The mechanism by which these stimuli activate HuR is unclear and might be related to post-translational modification of this protein.

The impaired polymerization of fibrinogen Longmont (Bbeta166Arg-->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers.

This study identified a new substitution in the Bbeta chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers.

Antifibrinogen IgG, fibrinogen, and Clq complexes circulating in a hypodysfibrinogenemic proband. Isolation, stoichiometry, and partial characterization.

Circulating antifibrinogen antibodies have been reported in rare afibrinogenemic propositi, apparently occurring following fibrinogen replacement therapy, but immune complexes have not been described. In this report we describe circulating immune complexes formed by a monoclonal antifibrinogen IgG in a heterozygous hypodysfibrinogenemic (A alpha 16 Arg-->Cys) proband. Estimated by partial protein sequence and by other analyses, each immune complex consisted of one fibrin(ogen), one C1q, and 3-4 IgG molecules.

Fibrinogen non-inherited heterogeneity and its relationship to function in health and disease.

In healthy individuals fibrinogen occurs in more than one million non-identical forms because of the many possible combinations of biosynthetically or postbiosynthetically modified or genetically polymorphic sites. The various forms may show considerable differences in their functional properties. Normal variant sites are due to alternative splicing, modification of certain amino acid residues, and proteolysis.

Modified clotting properties of fibrinogen in the presence of acetylsalicylic acid in a purified system.

To assess how treatment with acetylsalicylic acid (ASA) alters the fibrin network structure, clotting was initiated in purified fibrinogen incubated with ASA by adding thrombin. Clotting time and maximum absorbance of the fibrin aggregation curve were used to demonstrate the potential of fibrin generation. The results showed that the clotting properties of fibrinogen decreased and that the affinity of plasminogen to fibrin or thrombin inhibition by antithrombin increased if plasminogen or antithrombin, respectively, were present in the reaction system.

Fibrinogen Longmont. A heterozygous abnormal fibrinogen with B beta Arg-166 to Cys substitution associated with defective fibrin polymerization.

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers.

Methylation of histone H3 by coactivator-associated arginine methyltransferase 1.

The preferential in vitro methylation of histone H3 by coactivator-associated arginine methyltransferase 1 (CARM1) has been proposed as a basis for its ability to enhance gene transcription [Chen, D., et al. (1999) Science 284, 2174-2177].

On the identification of beneficial and detrimental molecular forms of fibrinogen.

Fibrinogen is a central protein in blood coagulation. A functioning circulation system requires a precise balance between fibrin formation and removal, i.e.

The crystal structure of modified bovine fibrinogen.

Here we report the crystal structure at approximately 4-A resolution of a selectively proteolyzed bovine fibrinogen. This key component in hemostasis is an elongated 340-kDa glycoprotein in the plasma that upon activation by thrombin self-assembles to form the fibrin clot. The crystals are unusual because they are made up of end-to-end bonded molecules that form flexible filaments.

Digestion of C1q collagen-like domain with MMPs-1,-2,-3, and -9 further defines the sequence involved in the stimulation of neutrophil superoxide production.

C1q, a subunit of the first component (C1) of the classical complement pathway, binds to neutrophils via its collagen-like region (C1q-CLR) stimulating superoxide production. We previously identified a region of C1q-CLR, defined by fragments generated by trypsin and endoLys-C digestion, that was required for triggering this respiratory burst. To further localize that critical site, purified human C1q was digested with pepsin to generate C1q-CLR, and subsequently cleaved with the matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9.

Crotalase, a fibrinogen-clotting snake venom enzyme: primary structure and evidence for a fibrinogen recognition exosite different from thrombin.

Crotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence [see text] characteristic of a serine proteinase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin's fibrinogen-recognition exosite (FRE) is poorly represented in crotalase.

Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerization, albeit more subtle than anticipated from the analogous proteolytic fragment X.

Numerous experiments have demonstrated that the C-terminal domain of the fibrinogen Aalpha-chain, the alphaC domain, has a role in polymerization. To further examine the role of this domain, we synthesized a recombinant fibrinogen, Aalpha251 fibrinogen, that lacks the alphaC domain. We examined thrombin-catalyzed fibrinopeptide release and found that the rate of FpB release from Aalpha251 fibrinogen was 2.

Structural examination of the influence of phosphorylation on the binding of fibrinopeptide A to bovine thrombin.

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood.

cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro.

The complement protein C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) are structurally similar molecules that enhance phagocytic function in vitro. Monoclonal antibodies R3 and R139, which inhibit the enhancement triggered by these three ligands, were used to purify a 126,000 M(r) cell surface protein designated C1qR(P). Amino acid sequence was obtained and the corresponding cDNA was cloned.

Photo-oxidation of histidine as a probe for aminoterminal conformational changes during fibrinogen-fibrin conversion.

Fibrinogen is known to become unclottable when irradiated with light in the presence of methylene blue, the loss of clottability being due to photo-oxidation of the histidine at position 16 of the B beta chain. In the present investigation it could be demonstrated that not only this histidine but also the one at position 24 of the A alpha chain was modified and that the rates of modification could be modulated by fibrinopeptide release, polymerization inhibition and denaturation. Accordingly, the histidine modifications can be used as probes for surface accessibility of and conformational differences among the various forms of the protein.

Localization of the site on the complement component C1q required for the stimulation of neutrophil superoxide production.

C1q, the recognition subunit of the classical complement pathway, interacts with specific cell surface molecules via its collagen-like region (C1q-CLR). This binding of C1q to neutrophils triggers the generation of toxic oxygen species. To identify the site on C1q that interacts with the neutrophil C1q receptor, C1q was isolated, digested with pepsin to produce C1q-CLR, and further cleaved with either trypsin or endoproteinase Lys-C.