Analysis of the mechanism, rate, and sites of proteolytic cleavage of human immunoglobulin D by high-pressure liquid chromatography.

Abstract

The high susceptibility of human immunoglobulin D to proteolytic degradation affects its biological function, metabolism, and immunoassay. High-pressure liquid chromatography was used to investigate the mechanism and rate of limited proteolytic cleavage of IgD and also to identify, isolate, and quantify the reaction products. Within 1 to 5 min, tryptic digestion of native IgD almost quantitatively yields a labile Fab fragment, a stable Fc fragment, and a highly charged peptide derived from the hinge region. A galactosamine-rich glycopeptide from the hinge region increases inversely as the Fab is largely degraded to a series of peptides within 1 hr. In contrast, the Fc and the high-charge peptide resist proteolysis for more than 24 hr. The initial sites of cleavage of IgD occur in the hinge region at exposed secondary structures predicted to be (-turns. Concomitant with removal of the galactosaminerich glycopeptide at its carboxyl terminus, the Fd fragment is rapidly and rather randomly degraded, but the light chain is somewhat more resistant than the Fd section of the 8 heavy chain. This study of the rapid rate of proteolysis ofIgD explains the rarity with which intact IgD is found in human sera. It also raises questions about immunoassay of IgD, which is usually measured with antisera against Fc. In vivo, proteolytic cleavage initiates the catabolism of circulating IgD and also affects the role and fate of IgD as an antigen receptor on the B-cell membrane.Unlike other immunoglobulins, human IgD has been little studied because its low concentration in normal serum and its great susceptibility to proteolytic degradation make it difficult to purify intact IgD for structural study. Nonetheless, the complete amino acid sequence of a human myeloma IgD protein has been determined in our laboratory (1)(2)(3) MATERIALS AND METHODS Materials. Human IgD was prepared from the plasma of patient WAH as described (7,8). In some experiments a second IgD, designated HUD, was used. The sources of trypsin and other enzymes and reagents (1,3,8) and of equipment, columns, and reagents for HPLC (9, 10) have been reported.Methods. Methods used in our laboratory for the determination of the primary structure of proteins and for their physical and immunochemical characterization (1,3,(7)(8)(9)(10) and the methods for computer analysis of sequence data (11) and for HPLC (9, 10) have been described.Most experiments on limited digestion were done under nondenaturing conditions with the WAH IgD and trypsin at an enzyme-to-substrate weight ratio of 1:100 in 0.1 M Tris HCl or 0.1 M ammonium bicarbonate (pH 8.0) at 37°C. In other experiments the temperatures ranged from 4°to 50°C. Digestion was stopped by addition of soybean trypsin inhibitor. In kinetic experiments the digestion times varied from 3 sec to 24 hr. In experiments with other proteolytic enzymes such as pepsin, plasmin, and kallikrein, conditions such as the pH and time of incubation were changed as seemed appropriate. Each digest was analyzed by NaDod-S04/PAGE (12-30% gr...

Differential Segmental Flexibility and Reach Dictate the Antigen Binding Mode of Chimeric IgD and IgM: Implications for the Function of the B Cell Receptor

“…To compute molar concentrations of IgD, IgD⌬h1, and IgMC575S, an accurate molecular mass determination was conducted by MS. The molecular mass of IgD and IgMC575S was estimated to 174.1 and 190.8 kDa, respectively, which are in agreement with previously published results (43)(44)(45). As for IgD⌬h1, it was found to be 158.8 kDa, which is 15.3 kDa less than the wt molecule.…”

Isolation and Characterization of a Novel IgD-Binding Protein fromMoraxella catarrhalis

“…In a study of the sera from ϳ50 myeloma patients, fragmented IgD was present in all cases, and usually this was the predominant form. In a study from Putnam's laboratory (49), high pressure liquid chromatography was used to investigate the mechanism and rate of limited proteolytic cleavage of IgD and also to identify and quantify the reaction products. Within 1-5 min, tryptic digestion of native IgD almost quantitatively yields a labile Fab, a stable Fc fragment, and a highly charged peptide from the hinge region.…”

Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco

“…T-DNA acts as an endogenous stimulus that activates the cellular machinery ( Fagard and Vaucheret, 2000 ). As a result, a previously stable genome can become particularly reactive in response to newly inserted transgenes, depending on the extent of inter-genic reactions ( Jones et al ., 1985 ; Gheysen et al , 1987 ; Mayerhofer et al , 1991 ; Petrov, 1997 ; Drews and Yadegari, 2002 ; Brunaud et al , 2002 ; Van Attikum and Hooykaas, 2003 ).…”

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