An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin.

Immunoassay technology is routinely used to measure concentrations of proteins and polypeptides in biological matrices. Increasingly, research efforts have sought to create analogs of human proteins with the aim of improving efficacy or pharmaceutical properties relative to the native protein. Pharmacokinetic assessment of these polypeptide analogs, however, can be greatly confounded by the presence of endogenous native protein. This report describes an immunization and immunoabsorption strategy that was used to create monospecific polyclonal antibodies against analogs of human leptin (LY355101 and LY396623, one and two amino acid changes relative to native human leptin, respectively). Rabbits were immunized with either LY355101 or LY396623. Antisera were screened to determine if any showed increased specificity for the analog relative to native human leptin. Antisera showing increased specificity for the leptin analog were then treated by immunoabsorption against native human leptin, thus depleting human leptin cross-reactivity. The antibodies developed in this process were used in radioimmunoassays. which were validated for use in clinical studies. Both assays proved to be highly specific for LY355101 or LY396623 in the presence of native human leptin. Use of this procedure permitted the measurement of LY355101 and LY396623 pharmacokinetics that were not confounded by the high levels of endogenous human leptin found in obese subjects. This technique has the potential for broad application in the development of assays capable of specifically measuring protein analogs without cross-reactivity to an endogenous substance.

Elastic fibre assembly: macromolecular interactions.

To investigate the mechanisms behind elastic fibre assembly, we studied the molecular interactions between elastin and microfibrillar components using solid-phase binding assays. Fibrillin 1, purified from tissue using reductive-saline extraction, showed no binding to microfibril-associated glycoprotein (MAGP) or tropoelastin. MAGP, however, was found to bind specifically to tropoelastin in a divalent-cation independent manner. Antibody inhibition studies indicated that the C-terminus of tropoelastin defined the interactive site with MAGP. MAGP and fibrillin were also substrates for transglutaminase, which may provide an important mechanism for stabilizing microfibrillar structure. In other studies we found that a major cross-linking region in elastin is formed through the association of domains encoded by exons 10, 19 and 25 of tropoelastin and that the three chains are joined together by one desmosine and two lysinonorleucine cross-links.

Identification of an elastin cross-linking domain that joins three peptide chains. Possible role in nucleated assembly.

The alignment of elastin molecules in the mature elastic fiber was investigated by purifying and sequencing cross-link-containing peptides generated by proteolytic digestion incompletely cross-linked insoluble elastin. Peptides of interest were purified by reverse phase and size exclusion high performance liquid chromatography and characterized by amino acid analysis and protein sequencing. One peptide, consisting of the cross-linking domain encoded by exon 10, contained a modified lysine residue that had not condensed to form a polyfunctional cross-link. Although this domain contains the characteristic paired lysine residues found in other cross-linking domains of elastin, protein sequence analysis indicated that the first but not the second lysine had been oxidized by lysyl oxidase. This finding suggests that lysine residues in an individual cross-linking domain may not have equal susceptibility to oxidation by lysyl oxidase. In a second peptide, we found that a major cross-linking site in elastin is formed through the association of sequences encoded by exons 10, 19, and 25 and that the three chains are joined together by one desmosine and two lysinonorleucine cross-links. Past structural studies and computer modeling predict that domains 19 and 25 are linked by a desmosine cross-link, while domain 10 bridges domains 19 and 25 through lysinonorleucine cross-links. These findings, together with the high degree of sequence conservation for these three domains, suggest an important function for these regions of the molecule, possibly nucleating the aggregation and polymerization of tropoelastin monomers in the developing elastic fiber.

Functional domains on elastin and microfibril-associated glycoprotein involved in elastic fibre assembly.

Studies in vitro suggest that the C-terminus of tropoelastin mediates elastin polymerization through an interaction with microfibril-associated proteins. In this study we have used cultured auricular chondrocytes as a model system to examine whether this interaction is critical for elastic fibre formation in vivo. Auricular chondrocytes, which deposit an abundant elastic fibre matrix, were cultured in the presence of Fab fragments of antibodies directed against the C-terminus (CTe) or an N-terminal domain (ATe) of tropoelastin. Immunofluorescent staining of the extracellular matrix deposited by the cells showed that the CTe antibody inhibited the deposition of elastin without affecting microfibril structure. Cells grown under identical conditions in the presence of ATe, however, formed fibres that stained normally for both elastin and microfibril proteins. Chondrocytes cultured in the presence of microfibril-associated glycoprotein (MAGP):21-35, an antibody directed against a domain near the N-terminus of MAGP, did not organize tropoelastin into fibres. However, immunostaining for MAGP and fibrillin revealed normal microfibrils. In agreement with the immunofluorescence staining patterns, fewer elastin-specific cross-links, indicative of insoluble elastin, were detected in the extracellular matrix of cells cultured in the presence of CTe. The medium from these cultures, however, contained more soluble elastin, consistent with an antibody-induced alteration of elastin assembly but not its synthesis. Northern analysis of antibody-treated and control cultures substantiated equivalent levels of tropoelastin mRNA. These results confirm that the C-terminus of tropoelastin interacts with microfibrils during the assembly of elastic fibres. Further, the results suggest that the interaction between tropoelastin and microfibrils might be mediated by a domain involving the N-terminal half of MAGP.

Biosynthesis of surfactant protein D. Contributions of conserved NH2-terminal cysteine residues and collagen helix formation to assembly and secretion.

Surfactant protein D (SP-D) is preferentially secreted as dodecamers consisting of four collagenous trimers cross-linked by disulfide bonds. In these studies, we examined the biosynthesis of wild-type rat SP-D (RrSP-D) and selected mutants by stably transfected CHO-K1 cells to determine the roles of a conserved N-linked oligosaccharide, the collagen helix, and interchain disulfide bonds in SP-D assembly and secretion. The major intracellular form of RrSP-D accumulated in the RER as complexes containing up to four trimeric subunits. Disulfide cross-link formation and RrSP-D secretion were selectively inhibited by 2,2'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylase, and by 2 mM dithiothreitol, but unaffected by tunicamycin or elimination of the consensus sequence for glycosylation at Asn70. Although mutants with serine substituted for Cys15 and Cys20 (RrSP-Dser15/20) are secreted as trimeric subunits, proteins with single cysteine substitutions were retained in the cell. Surprisingly, the secretion of RrSP-Dser15/20 was unaffected by 2,2'-dipyridyl. These studies strongly suggest that the most important and rate-limiting step for the secretion of SP-D involves the association of cross-linked trimeric subunits to form dodecamers stabilized by specific inter-subunit disulfide cross-links. Interference with collagen helix formation prevents secretion by interfering with efficient disulfide cross-linking of the NH2-terminal domain.

Site-directed mutagenesis of Cys-15 and Cys-20 of pulmonary surfactant protein D. Expression of a trimeric protein with altered anti-viral properties.

Surfactant protein D (SP-D) molecules are preferentially assembled as dodecamers consisting of trimeric subunits associated at their amino termini. The NH2-terminal sequence of each monomer contains two conserved cysteine residues, which participate in interchain disulfide bonds. In order to study the roles of these residues in SP-D assembly and function, we employed site-directed mutagenesis to substitute serine for cysteine 15 and 20 in recombinant rat SP-D (RrSP-D), and have expressed the mutant (RrSP-Dser15/20) in Chinese hamster ovary (CHO-K1) cells. The mutant, which was efficiently secreted, bound to maltosyl-agarose, but unlike RrSP-D, was assembled exclusively as trimers. The constituent monomers showed a decreased mobility on SDS-polyacrylamide gel electrophoresis resulting from an increase in the size and sialylation of the N-linked oligosaccharide at Asn-70. Although RrSP-Dser15/20 contained a pepsin-resistant triple helical domain, it showed a decreased Tm, and acquired susceptibility to proteolytic degradation. Like RrSP-D, RrSP-Dser15/20 bound to the hemagglutinin of influenza A. However, it showed no viral aggregation and did not enhance the binding of influenza A to neutrophils (PMN), augment PMN respiratory burst, or protect PMNs from deactivation. These studies indicate that amino-terminal disulfides are required to stabilize dodecamers, and support our hypothesis that the oligomerization of trimeric subunits contributes to the anti-microbial properties of SP-D.

Evidence that enviroxime targets multiple components of the rhinovirus 14 replication complex.

Enviroxime and related analogs are potent inhibitors of rhinoviruses and enteroviruses in cell culture. Previous analyses of resistant mutants implicated the viral nonstructural protein 3A(B) as the likely target of drug activity. In this study, we used site-directed mutagenesis and selection of spontaneous rhinovirus 14 mutants with several enviroxime analogs to confirm the link between domains in rhinovirus 14 3A(B) and the function blocked by enviroxime. We also produced recombinant 3A and 3AB proteins for biochemical analyses. Despite extensive efforts, however, we were unable to demonstrate direct binding between enviroxime and any of several viral proteins, nor could we demonstrate binding of enviroxime to a host protein. In addition, enviroxime did not disrupt 3AB's ability to bind RNA or 3D polypeptide, the association of 3AB with membranes, or the cleavage of 3AB by 3C protease. Finally, we identified an enviroxime-resistant mutant with an increased level of resistance which apparently has mutations in multiple proteins or RNA sequences. Taken together, these results suggest that enviroxime targets a complex of proteins and/or cellular factors. Such a complex mechanism of inhibition might explain the low levels of viral resistance to these inhibitors as compared with other picornaviral inhibitors.

Microfibril-associated glycoprotein binds to the carboxyl-terminal domain of tropoelastin and is a substrate for transglutaminase.

Microfibril-associated glycoprotein (MAGP) is an integral component of microfibrillar structures that play a critical role in the organization of elastic fibers in the extracellular matrix. To study possible molecular interactions between MAGP and other elastic fiber components, we have generated native MAGP using a baculovirus expression system and tested its ability to associate with tropoelastin and fibrillin. MAGP produced by SF9 cells underwent processing similar to the mammalian protein, including correct cleavage of the signal peptide and sulfation of tyrosine residues. When tested in solid-phase binding assays, native MAGP specifically bound to tropoelastin but not fibrillin-1. Binding to tropoelastin was divalent cation-independent and was completely blocked by reduction and alkylation of either protein. Antibody inhibition studies indicated that the carboxyl terminus of tropoelastin mediated its interaction with MAGP. In addition to binding to elastin, MAGP was also a substrate for transglutaminase, which might explain its propensity to form high molecular weight aggregates that cannot be dissociated with reduction or denaturation. Together, the results of this study provide new insights into the functional relationship between microfibrillar proteins and have important implications for understanding elastic fiber assembly.

Sensitive RIA for the specific determination of insulin lispro.

Insulin lispro is an insulin analog in which the primary sequence has been altered by the inversion of amino acids B28 and B29. To date, it has not been possible to specifically measure insulin lispro in the presence of endogenous insulin because of the high degree of homology between these peptides. However, the specific determination of insulin lispro offers advantages over quantifying total concentrations of immunoreactive insulin. We therefore immunized guinea pigs and screened for antibodies with increased affinity and selectivity for insulin lispro. We prepared a monospecific antiserum by a novel immunoadsorption strategy using despentapeptide insulin. The antiserum was used to develop a competitive RIA for insulin lispro. The RIA has a low limit of quantification (17.2 pmol/L); has no interference from insulin, proinsulin, or C-peptide; and has interassay CVs of 2.6-13.4%. The new RIA is useful for measuring serum concentrations of insulin lispro.