One-step sandwich enzyme immunoassays for human C4b-binding protein (C4BP) and protein S-C4BP complex using monoclonal antibodies.

C4b-binding protein (C4BP), a regulatory component in the complement system, binds to an anticoagulant vitamin K-dependent plasma protein S (PS) which acts as a cofactor of activated protein C. We raised monoclonal antibodies against C4BP and PS, and developed two different one-step sandwich enzyme immunoassay (EIA) systems for human total C4BP (assay A) and PS-C4BP complex (assay B) by using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The reaction time of the assay was 45 min in both EIA systems: 30 min for the immunoreaction and 15 min for the color reaction. The sensitivities were 12 and 20 mg/l in assays A and B, respectively. Linearity was obtained between 31 and 500 mg/l in both EIA systems. Assay A could detect both uncomplexed C4BP and PS-C4BP complex with equal efficiency so that total C4BP level was not affected by PS. The levels of total C4BP and PS-C4BP complex were found to significantly increase in sera from patients with membranous nephropathy and decrease with liver cirrhosis in comparison with the levels in normal subjects. On the other hand, a difference in the total C4BP and PS-C4BP complex levels was not shown between IgA nephropathy and normal subjects. Affinity column analysis and difference of total C4BP and PS-C4BP complex levels showed that most of C4BP in sera exists as PS-C4BP complex.

Monoclonal antibodies against two epitopes in the human alpha 1 (IX) collagen chain.

Type IX collagen is a component of cartilage and vitreous humor. Its structure and matrix localization suggest it may serve to mediate interactions between fibrillar collagen, proteoglycan and other matrix components. Consequently, abnormalities in type IX collagen may result in chondrodysplasia. In this paper we describe the preparation and use of two monoclonal antibodies which recognize peptide sequences within the human cartilage alpha 1 (IX) collagen chain. Antibody 23-5D1 is highly sensitive and highly specific. It permits the immunoblot detection of type IX collagen extracted from milligram amounts of normal and chondrodysplastic cartilage; it also identifies the "short" form of the alpha 1 (IX) chain in human vitreous humor. Antibody 37-10H7 is highly specific, but of low sensitivity. It was used to make the new observation that an N-linked oligosaccharide is present in the amino-terminal globular domain of the alpha 1 (IX) chain. We anticipate that these antibodies may be valuable tools in the study of human and other mammalian chondrodysplasias.

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.

The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats.

Human plasma carboxypeptidase N is a 280-kDa tetramer with two high molecular mass (83-kDa) glycosylated subunits which protect the two 50-kDa catalytic subunits and keep them in the circulation. An initial clone for the 83-kDa subunit was obtained by screening two lambda gt11 human liver cDNA expression libraries with antiserum specific for carboxypeptidase N or the 83-kDa subunit. The libraries were rescreened with the labeled cloned cDNA, and the largest clone obtained (2536-base pair insert) was completely sequenced. The deduced protein sequence matched the sequence of several tryptic peptides from the 83-kDa subunit but did not contain the NH2-terminal sequence. The remaining portion of the protein coding sequence was synthesized by the polymerase chain reaction, cloned, and sequenced. The composite cDNA sequence is 2870 base pairs long with an open reading frame of 1608 base pair coding for a protein of 536 amino acids (Mr = 58,762). The protein sequence contains seven potential N-linked glycosylation sites and a threonine/serine-rich region which is a potential site for attachment of O-linked carbohydrate. The most striking feature is a region (residues 68-355) containing 12 leucine-rich tandem repeats of 24 residues with the following consensus sequence: P-X-X-alpha-F-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-L-X-X-L (X = any amino acid and alpha = aliphatic amino acids, I, L, or V). This repeating pattern is found in the leucine-rich alpha 2-glycoprotein and in other proteins where it might mediate interactions with macromolecules. This region also contains five sequences with heptad repeating leucine residues comprising a leucine zipper motif. The leucine-rich domain likely constitutes an important structural or functional element in the interactions of the 83- and 50-kDa subunits to form the active tetramer of carboxypeptidase N.