W. Manski

Immunochemical studies on albuminoid.

Albuminoid of the adult lens contains in addition to “insolubilized” α-crystallin, pre-α β- and γ-crystallins. There is a difference in their quantitative distribution among the cortical and nuclear albuminoid. The latter contains more of the β- and γ-antigens. In both, α-crystallin is the major constituent. Cross-absorption or immunodiffusion tests did not reveal loss of antigenic determinants present in lens crystallins or appearance of new determinants in albuminoid. Albuminoid formation thus appears to be a progressive process that starts with conformational changes of α-crystallin (possibly together with one of the β and/or pre-α molecules). The “insolubilization” in time involves to varying degrees the majority, if not all, lens crystallins.

Immunochemical studies on lens protein-protein complexes I. The heterogeneity and structure of complexed α-crystallin

Abstract A buffered extract of young cattle lenses filtered through Diaflo XM-300 membranes, retaining molecules above 300 000 daltons, was found to contain in the non-filterable fraction constituting 50% of soluble lens proteins α-and β-crystallins. The non-filterable fraction of soluble proteins from mature cattle lenses, constituting 87% of all crystallins, contained not only α- and β-crystallins, but γ-crystallins as well. Only α-crystallin, with a molecular weight above 300 000 daltons, was expected to remain in the non-filterable fraction. All of the β-crystallins, which have a molecular weight below 200 000 daltons, and γ-crystallins, with a molecular weight of about 20 000 daltons, if free in solution and not complexed, were expected to be in the filtrate. The lens proteins with a molecular weight above 300 000 daltons from young lens tissue were separated by gel chromatography (Bio-Rad A 15 m) into free α-crystallin followed by an α-β-crystallin complex. By the same procedure, the proteins above 300 000 daltons from mature lenses were separated into two complexes of quantitatively different α-β-γ-crystallin composition. A dependence of lens protein complex formation on the ionic composition of the solvent was observed. The differences between calf cortical and bovine nuclear complexes, found in buffer extracts, were much less pronounced in water extracts in which both contained γ-crystallins. Specific dissociation of the lens protein complexes was obtained by use of an anti-α-crystallin immunoadsorbent, as shown by the passage of the previously retained β- and γ-crystallins through a Diaflo XM-300 membrane. An anti-β-crystallin immunoadsorbent reacting with complexes from mature lens tissue yielded a non-filterable α-crystallin and a filterable γ-crystallin in the supernatant, indicating an absence of direct α-γ complexes. These results point to a potentially central role of β-crystallins in complex formation among lens proteins.

Immunochemical studies on albuminoid: II. Changes associated with age☆

Abstract Immunochemical investigations of albuminoid from young and adult cattle lenses showed that the composition of albuminoid is age-dependent. An increase in the β, as well as a decrease of the pre-α-crystallin antigen reaction was observed. The γ-crystallin reaction declined in the adult cortical albuminoid, then increased again in the adult nuclear albuminoid but to a level still lower than in calf albuminoid. The least difference with age was observed for the α-crystallin antigen. The increase in the β-crystallin reaction with time appeared to be the result of an increased participation of some minor β antigens in the formation of albuminoid. This increase did not involve the main β antigen in albuminoid, which constitutes approximately 3% of the insoluble fraction. The decreased pre-α reaction in the adult albuminoids indicates that this crystallin antigen may have a limited range of structural states at which it is able to participate in the formation of albuminoid. The initial decrease and subsequent increase with age in the γ-crystallin reaction in albuminoids points to the possibility that a minimum of two γ antigens are involved in the formation of albuminoid at different kinetic rates.

Immunochemical studies on lens protein-protein complexes II. On the mechanism of alpha-beta-gamma crystallin complex formation

Abstract An analysis of the β- and γ-crystallins present in various lens protein complexes of α-β, α-β-γ or only β-γ crystallins was done. This analysis was based on a detailed study of different proteins in the β- and γ-crystallin fractions. Beta and γ-crystallins were isolated from the different complexes by reacting each of them with anti-β crystallin antibodies used as ligands in an immunoadsorbent. The reaction resulted in dissociation of the complexes and binding of the various β-crystallins to the immunoadsorbent, leaving α- and γ-crystallins free in solution. After elution and concentration, the β-crystallins were compared in immunodiffusion tests with the high and low molecular weight β-crystallins. The freed γ-crystallins were first separated from α-crystallin by filtration on Diaflo XM-300 membranes. The filtrates were concentrated and compared with high and low molecular weight γ-crystallins. All of the β-crystallins which complexed with α-crystallin to form the α-β as well as the different α-β-γ crystallin complexes were found to contain β-crystallin molecules I and II, molecules which are typical of high molecular weight β-crystallin fractions. In addition, none of these β-crystallins contained β-crystallin molecule VII which is typically found in low molecular weight β-crystallin fractions. By contrast, all of the β-crystallins which did not complex with α-crystallin but which did form various β-γ crystallin complexes were related to the low molecular weight β-crystallins, as shown by the presence of β-crystallin molecule VII in these complexes and the absence of β-crystallins I and II. β-Crystallin molecules III through VI present in both high and low molecular weight β-crystallin fractions were found in all complexed crystallins. All complexes were also found to contain the same γ-crystallin molecules I and II. The data indicate that with progressing lens age, an initial α-β complex binds free γ-crystallins forming α-β-γ crystallin complexes. The latter complexes are not formed by association of α-β with β-γ complexes.

Immunochemical analysis of tissue antigens in different corneal layers and sclera

Abstract Tissue-specific antigens were studied in cornea and sclera with antisera absorbed with serum proteins. Both human and bovine corneal tissue-specific antigens were able to elicit a much stronger antibody response than scleral antigens. The presence of antigens, restricted only to individual corneal tissues, was established. Among the tissue antigens shared by different corneal tissues some were found to be common to all. Four antigens were shown to be shared by stroma and endothelium only, whereas one antigen was shared by stroma and epithelium. Thus, common ontogeny did not seem to be the only determining factor in the antigenic relation of different corneal tissues. Cornea and sclera were found to share antigens. These were restricted to the stromal layer. The anti-bovine corneal epithelium serum gave no precipitin reactions with sclera. Characteristic differences in the electrophoretic behavior of tissue-specific antigens from different corneal layers were observed. In agar immunoelectrophoresis the bovine epithelial antigens showed low electronegative net charge, while the endothelial antigens exhibited high electronegative net charge. The stromal antigens were evenly distributed along the electrophoretic path. The observed net charge differences are discussed in terms of the greater ion-binding capacity of endothelial vs. epithelial protoplasmic macromolecules.

Comparison of α-Crystallin A and B chains in their dissociated and associated states

Changes occurring during the heterologous association of A with B chains to form α-crystallin molecules were compared to changes occurring during homologous association of these chains to form aggregate molecules of A with A or B with B chains. This analysis of the α-crystallin subunit chains was done with antibodies to dissociated A or B chains and with antibodies restricted to each of these chains isolated from an immune serum to α-crystallin in which the A and B chains were associated with each other. In separated, aggregated A chains, only about 25% of the antigenic determinants were found to be the same as those which occur in the monomeric A chains. In the aggregated B chains, about 58% of the determinants were found to be also reactive in the monomeric B chains. These findings indicate that significantly greater conformational changes take place during the aggregation of A than of B chains. Antigenically, the A and B chains were found to be completely different. The antigenic determinants of A chains in α-crystallin constitute 92% of all of the antigenic determinants in separated, aggregated A chains. The 8% of determinants present in separated, aggregated A chains which were non-reactive in α-crystallin or absent from it were also nonreactive in monomeric A chains; thus, these determinants must be aggregation-dependent. The antigenic determinants of A chains in α-crystallin and those of separated, aggregated A chains showed the same ratio of monomeric to aggregation-dependent antigenic determinants. The B chains in α-crystallin and in the separated, aggregated B chains were found to be antigenically identical. These results are compatible with the view that early post-translational changes of A and B chains may first involve homologous aggregation of each chain, followed by conformational changes which lead to the association of A with B chains in a 2:1 ratio. An association of two monomeric A chains with one monomeric B chain, e.g. leading first to the formation of a “primary” α-crystallin molecule which then aggregates further, could be expected to yield different immunochemical data. This hypothesis is under further investigation.

An immunochemical study of the different proteins in the beta and gamma crystallin families

Abstract The crystallins isolated from calf lens cortex were separated by gel filtration on Bio-gel A 0·5 m columns. Immunoelectrophoretic and immunodiffusion analysis of these crystallins demonstrated complete separation of the α-crystallins (fraction 1), various β-crystallins (fractions 2, 3 and 4) and γ-crystallins (fractions 5 and 6). The presence of at least seven different β-crystallin proteins (in terms of different genetic products) were established. The high molecular weight β-crystallins (fraction 2) contained proteins I and II, which were absent from the low molecular weight β-crystallins (fraction 4). Conversely, the low molecular weight β-crystallins contained protein VII, which was absent from the high molecular weight fraction, β-Crystallins III, V and VI were present in both of these factions. The medium molecular weight β-crystallins (fraction 3) contained proteins III through VII. Both the high (fraction 5) and the low (fraction 6) molecular weight γ-crystallins contained proteins I and II. The use of Bio-gel A 0·5 m columns yielded better separation of α-from β-crystallins than did the Sephadex G-200 columns. This difference may have resulted from more efficient dissociation of α-β crystallin complexes on the Bio-gel A 0·5 m than on the Sephadex G-200.

Immunochemical studies on lens protein-protein complexes III. Age-related changes in the stability of lens alpha crystallin containing compleces

Abstract The age dependence of the stability of lens crystallin complexes was investigated. The lens protein fractions retained on Diaflo XM-300 membranes were subsequently separated on Bio-gel A 15 m columns, yielding one calf cortical α-β crystallin complex and two different bovine nuclear α-β-γ crystallin complexes. Separate samples of these different lens crystallin complexes were exposed for various lengths of time to acidic or alkaline pH, or to high concentrations of salts at neutral pH. Acidic pH, three units below the neutral, had a more pronounced dissociating effect on the crystallin complexes than alkaline pH, three units above the neutral, or a high salt concentration. The dissociation of the various complexes was tested by ultrafiltration on Diaflo XM-300 membranes and by immunochemical analysis of the fractions retained and filtered. Comparison of the calf cortical and bovine nuclear complex preparations indicated that the stability of the crystallin complexes increases with progressive aging of the lens. The crystallin complexes investigated did dissociate in a constant electrical field, yielding free α-, β- and γ-crystallin molecules.

II. Immunochemical studies on alpha crystallin subunits

Abstract Carboxymethylated alpha crystallin subunits yield a clearer separation of the acidic and basic subunit molecules than native alpha crystallin subunits. A minimum of four distinct subunit molecules could be observed in all fractionation procedures tested. The heterogeneity of these subunit molecules was more pronounced on DEAE-cellulose chromatography with slightly basic buffers than on SP-Sephadex with acidic buffers. The latter procedure yielded an immunochemically pure, acidic subunit molecule. The basic fraction included at least two different antigens. Two minor fractions, each containing the same two additional antigens, eluted in front of the acidic fraction. These latter antigens occurred in all DEAE-cellulose chromatographic fractions. It is stressed that the subunits separated in all these procedures were finally analyzed as reaggregates. In view of the occurrence of differently desorbed but antigenically equivalent subunit molecules the possibility is discussed that these reaggregated subunit molecules may be built from the same subunits of alpha crystallin but in different proportins and/or conformatinal states.

Alpha neoprotein molecules in normal lenses from animals of different ages and in cataractous lenses

Alpha neoprotein molecules are formed by an association of A and B subunits resulting in a different quaternary structure than in alpha crystallin. The content of alpha neoprotein vs. alpha crystallin was estimated separately on the crystallin, cryoprotein and albuminoid fractions in lenses of animals of different species and ages, in lenses with experimentally induced cataracts and in human cataractous lenses. Alpha crystallin was determined on immunoadsorbents with bound antibodies restricted to quaternary determinants of this protein. Alpha neoprotein molecules were determined in the filtrate containing lens proteins not bound on the above immunoadsorbent. The filtrate was applied on an anti-A chain immunoadsorbent, and after desorption the A chain-containing molecules were applied on an anti-B chain immunoadsorbent. The amount of lens proteins bound to the latter immunoadsorbent gave a measure of molecules formed by an association of A with B chains other than in alpha crystallin, that is of alpha neoproteins. No alpha neoprotein was present in lenses from 2-week-old rats or in the 3- to 6-month-old calf lens cortex. The 2-year-old bovine lens nucleus did contain alpha neoproteins, but only in the albuminoid fraction. The lenses of adult rats (i.e. 3- to 4-month-old) contained alpha neoprotein molecules mainly in the albuminoid fraction; small amounts were also found in the cryoprotein fraction. All three fractions of lens proteins from 1-year-old rats contained alpha neoprotein molecules. No significant differences in the content of alpha neoprotein molecules were found in lenses with experimentally induced X-ray and galactose cataracts and in normal controls. This finding does not exclude possible structural differences between normal and cataractous alpha neoproteins. In human senile cataractous lenses, the content of alpha neoprotein molecules in all fractions analyzed equaled or exceeded the content of alpha crystallin present. The present findings demonstrate an age-dependent formation in the mammalian lens of alpha crystallin neoproteins lacking native quaternary determinants. The data obtained point to the possibility that the structural lability of alpha crystallin may be a contributing factor in cataractogenesis.