Molecular basis for the temperature-dependent insolubility of cryoglobulins--XI. Sequence comparison of the heavy-chain variable regions of the human cryoimmunoglobulins McE and Hil by metric analysis.

The amino acid sequences of the VH-domains from two human cryoimmunoglobulins have been compared with one another and with the VH-domains of noncryoglobulins by the mathematical method of metric analysis. The VHII sequence of McE [Gerber-Jenson et al. (1981), J. Immun. 126, 1212-1216] and the VHIII sequence of Hil [Chiu et al (1979), Biochemistry, 18, 553-560] resemble much more closely the VH-sequences of noncryoglobulins from their own subgroups than they resemble one another. Neither cryoglobulin sequence contains an unusual insertion or deletion of residues. Based on the crystallographic structure of the VHII domain in the Fab fragment of the human noncryoglobulin Newm [Saul et al. (1978), J biol. Chem., 253, 585-597], McE and Hil each contain two unprecedented residues in the outer beta-sheet structure of the VH-domain. The inwardly directed sidechains of Gly-71 and Ile-84 in McE may perturb the internal hydrophobic interactions and normal folding of adjacent segments of the outer beta-sheet from the third framework region. In contrast, the outwardly directed sidechains of Ile-23 and Arg-77 in Hil may perturb the external hydrophilic properties and folding of adjacent segments of the outer beta-sheet from the first and third framework regions. Thus, the monoclonal immunoglobulins McE and Hil may display cold-induced insolubility by different perturbations of the outer surface of the VH-domain. In each case, the unprecedented framework residues that mnay be responsible could have arisen by two point mutations involving single base changes.

Lysines of histone 1 represent the principal target for covalent binding of microsomally activated benzo[a]pyrene in vitro.

The principal target amino acid residue for covalent binding of metabolically activated [3H]benzo[a]pyrene (B[a]P) to histone 1 (H1) has been identified. Highly purified calf thymus H1 was modified by incubation with [3H]B[a]P in the presence of rat liver microsomes. The relative distribution of [3H]B[a]P versus specific amino acids among the N-bromo-succinimide peptides of H1 suggested that lysine may be the target. This was tested by selectively blocking the nucleophilic amino groups of lysine by amidination prior to [3H]B[a]P binding. Increasing levels of amidination of H1 resulted in corresponding decreases in the reactivity of this histone toward [3H]B[a]P. Amidination of 93% of the lysines blocked 87% of the [3H]B[a]P binding indicating that lysine was the principal target. N-terminal amino acid sequencing of [3H]B[a]P-modified H1 confirmed that lysine residues had bound [3H]B[a]P.

Molecular basis for the temperature-dependent insolubility of cryoglobulins. X. The amino acid sequence of the heavy chain variable region of McE.

The amino acid sequence of the VH region of McE, a monoclonal IgM cryoimmunoglobulin, has been determined employing automated sequencing methodology. Digestion of the intact Fab mu component, derived by trypsin cleavage of the parent protein at elevated temperature with CNBr, followed by complete reduction and alkylation, yielded the intact light chain as well as the 2 CNBr fragments that constituted the VH. N-terminal sequencing of the larger unblocked CNBr fragment, along with a component fragment derived by cleavage by BNPS-Skatole, established the structure of the VH from position 88 through the V leads to C switch region. Citraconylation of the smaller, blocked fragment effected sufficient solubilization for enzymatic deblocking and direct sequencing of the N-terminal 20 residues of the VH. Complete trypsin digestion of the N-terminal CNBr fragment yielded 9 peptides that could be isolated by preparative cation exchange chromatography and gel filtration. The complete sequence of these peptides along with 4 chymotryptic peptides completed the primary structure of the VH region. The primary structure of McE appears to resemble that of He, previously identified as belonging to the VH II subgroup. The presence of characteristic CDR and FR regions as well as the identification of a probable site of glycosylation suggest that the cryoimmunoglobulin closely resembles noncryoglobulins in terms of overall structural composition. The cryoglobulin property may arise through alterations in individual residues or unfavorable arrangements of CDR and FR segments.

Molecular basis for the temperature-dependent insolubility of cryoglobulins. XII. Anomalous mobility of monoclonal cryoimmunoglobulin heavy chains accompanying polyacrylamide gel electrophoresis in sodium dodecyl sulfate.

When subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), the fully reduced and alkylated heavy chains isolated from three monoclonal IgG1 cryoimmunoglobulins exhibited various degrees of retardation in mobility when compared to noncryoglobulin references. The anomalous electrophoretic mobility was not correlated with the thermal magnitude of cryoprecipitation of the individual proteins. High sensitivity analytical gel filtration in 5 M guanidine-HCl failed to distinguish heavy chains of the cryoimmunoglobulins from noncryoglobulin references, suggesting that the proteins possess equivalent molecular weights. Other possible causes for the anomalous mobility such as atypical amino acid and carbohydrate composition, charge and quantitative SDS binding do not appear to be likely. It remains possible that the shape and/or charge of the SDS-protein complexes are unique. Examination of the gel electrophoretic mobility in SDS of fully reduced and alkylated Fab components suggests that the Fd portion of these proteins may be abnormal. The gel electrophoresis anomaly is the only atypical structural feature thus detected which is shared by these three monoclonal cryoimmunoglobulins.

Immunoglobulin diversity in the phylogenetically primitive shark, Heterodontus francisci. Suggested lack of structural variation between light chains isolated from different animals.

A two-step procedure employing gel filtration and anion exchange chromatography has been utilized to isolate LMW immunoglobulin from the horned shark, Heterodontus francisci. Light chains obtained by complete reduction and alkylation of the parent protein have been compared by several analytical techniques. Amino acid composition data implies a limited degree of variation in the light chains isolated from individual animals. Polyacrylamide gel electrophoresis of the CNBr digests of the light chains reveal indistinguishable banding profiles of the major peptides. Isoelectric focusing indicates limited heterogeneity in the light chain spectrotype and identity in the pI of the majority of bands detectable by staining. The suggested degree of structural similarity in the light chains of this phylogenetically primitive shark is discussed in terms of the evolutionary position of the species and current theories concerning the origins of structural diversity in immunoglobulins.

Structurally unique basic protein coextracted with histones from calf thymus chromatin.

A histone-like protein that is rich in alanine and lysine (protein AK) has been obtained in homogeneous form by high-resolution gel filtration of H2SO4 extracts of calf thymus chromatin. Protein AK: (i) migrates as a single band in polyacrylamide gel electrophoresis in both acetic acid/urea and sodium dodecyl sulfate; (ii) is a basic protein; (iii) lacks tryptophan; (iv) is not extracted from chromatin with 0.35 M NaCl; and (v) is not soluble in 0.75 M HClO4. Protein AK is distinguished from the high-mobility group (HMG) proteins on the basis of these latter solubility characteristics and from the histones and protein A24 on the basis of amino acid composition and distribution of tryptic peptides in two-dimensional chromato-electrophoresis. In addition, protein AK is distinguished from the HMG proteins, the histones, and protein A24 on the basis of its mobility in two polyacrylamide gel electrophoresis systems. The amino acid composition of protein AK resembles that of HU, a basic DNA-binding protein found in Escherichia coli.

Physicochemical characterization of six monoclonal cryoimmunoglobulins: possible basis for cold-dependent insolubility.

The physical and chemical properties of five human and one canine monoclonal cryoimmunoglobulin have been compared. By many criteria, the proteins cannot be distinguished from the noncryoglobulin reference proteins analyzed in parallel; however, certain hydrodynamic and spectroscopic properties of the proteins indicate that cryoimmunoglobulins differ in tertiary structure relative to their cold-soluble counterparts. These differences seem to favor low-temperature-induced association between cryoglobulin molecules as an immediate consequence of increased intermolecular ionic or van der Waals forces. No evidence was found for the formation of cold-dependent antigen-antibody complexes or the ubiquitous presence of low-temperature-dependent conformation changes as a component of cryoprecipitation. Rather, the anomalous solution behavior of monoclonal cryoimmunoglobulins can be considered a direct result of the individual solubility properties of these proteins.

The structure of immunoglobulin variable regions in the horned shark,Heterodontus francisci.

The heavy and light chains of pooled antibodies of the hybodont shark,Heterodontus francisci (horned shark), were subjected to amino acid sequence analysis. Yield determinations showed that more than 90% of the available polypeptides in the respective pools were sequenced. The heavy chains were homogeneous in the initial framework segment and showed a sequence homology of approximately 70% with the corresponding region of the more recently evolved nurse shark and a 45% homology with a human myeloma heavy chain. The light chains were less homogeneous and not identifiable as either kappa or lambda chains as known in higher species. The first half-cystine characteristics of the variable domain intrachain disulfide bridge of immunoglobulins was present in the same position (22 for heavy chains; 23 for light chains) in the horned shark as in mammalian species. The sequence analysis also suggested the presence of a hypervariable region in the horned shark light chains. The combined data imply that the antigen-binding function of immunoglobulins is mediated in much the same manner in this primitive shark as in more recently evolved species, including mammals.