Lens alpha-neoproteins.

The formation of lens alpha-neoprotein molecules which are built by the association of A and B subunit chains of alpha-crystallin but have a different quaternary structure than the native protein is a posttranslational process that progresses through the life span. Alpha-neoproteins occur regularly in all mature mammalian lenses tested. The mechanisms that have been identified for the formation of alpha neoproteins are imbalance in the biosynthesis of A- to B-chains, which leads to a ratio of less than 2A-chains to 1B-chain, and derivation of the chains after their initial association into native alpha-crystallin. In terms of quantity of neoproteins, no significant differences have been found between lenses with developing cataracts and normal control lenses of corresponding age. The possibility that there are structural differences between alpha-neoproteins in normal and cataractous lenses of the same age, as well as potential differences among alpha-neoproteins isolated from normal lenses of different ages, are presently under investigation.

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.

Modification of alpha-crystallin subunit chains and formation of alpha-neoprotein molecules: role of SH groups.

Immunoadsorbents with bound antibodies restricted to determinants dependent on alpha-crystallin's quaternary structure permitted the fractionation of the population of 125I-labeled alpha-crystallin molecules, treated by iodoacetic acid, into molecules in which the native structure was still preserved and molecules with a completely different quaternary structure than the native protein. Parallel experiments with [14C]iodoacetic acid yielded information on the percentage of blocked SH groups in each of the above two fractions. The presence of molecules formed by A with B-chain association was established by sequential binding first to an immunoadsorbent with antibodies restricted to determinants located on alpha-crystallin's A-subunit chains as ligand and second, after desorption, to an immunoadsorbent with antibodies to B chains as ligand. With the aid of these techniques, it was established that (i) The modified alpha-crystallin molecules with quaternary determinants of the native protein contained a maximum of 23% blocked SH groups, indicating that the carboxymethylation involved only the fast-reacting surface SH groups. (ii) The modified alpha-crystallin molecules without the native protein's quaternary structure were built by a different association between A and B subunits than in alpha-crystallin, indicating formation of alpha-neoprotein molecules. (iii) Monomeric A chains with all SH groups carboxymethylated, and monomeric B chains in a ratio of 1A:5B, 2A:1B, and 5A:1B in urea solution, associate on dialysis, forming alpha-neoprotein molecules.

Antigen-antibody reactions in the eye involving complement binding IgG antibodies and their non-complement binding F(ab')2 fragments.

An immunogenic inflammation was induced in the eyes of inbred Wistar-Furth rats by intravenous injection of anti-human serum albumin IgG antibodies and intravitreal injection of human serum albumin. The ocular inflammation was compared to the findings observed following the intravenous injection of F(ab')2 fragments of anti-human serum albumin IgG antibody and intravitreal injection of human serum albumin. The dose of antigen and IgG immunoglobulin antibody molecules used in the experiments caused a consistent inflammatory response that reached a peak at 24 hours and lasted for approximately seven days. The experimental animals, in which an intraocular reaction of the antigen with F(ab')2 antibody fragments occurred, did not show any immunogenic inflammatory response, indicating an absence of complement activation by either the classical or the alternative pathways.

Interaction between the constituent A and B lens alpha-crystallin subunits leading to formation of alpha-neoprotein molecules.

A and B constituent subunits associated in lens alpha-crystallin were found to interact with added B chains forming alpha-neoprotein molecules with lower A to B chain ratios than 2 A to 1 B in alpha-crystallin. Addition of 1% excess of B chains to the one in alpha-crystallin, which resulted in a ratio of 1.98 A to 1 B in the mixture, caused a change of quaternary structure in 30% of alpha-crystallin molecules within 18 h. At a ratio of 1.86 A to 1 B, all alpha-crystallin molecules were affected at this time. A maximum number of 495 B chains was found to form an association with 1 A chain, initially bound in alpha-crystallin. Such a high number may indicate that the reaction involves monomeric A chains binding aggregated macromolecules of B chains. It is in such form that B chains occur as macromolecules with an average molecular weight of 0.7 X 10(6) in aqueous solution. The alpha-neoprotein molecules selected for studies in this report had A to B chain ratios of 1.75:1, 1:1, and 0.2:1. Each behaved in immunodiffusion tests like single molecular entities. Antigenic determinants located on A as well as on B chains associated with each other in alpha-crystallin were found to be identical with determinants on the chains associated in the above alpha-neoprotein molecules. Determinants dependent on the quaternary structure of alpha-neoprotein and of alpha-crystallin molecules were completely different. Some of the quaternary determinants of various alpha-neoproteins were type specific and did not occur in molecules with different A to B chain ratios. Other quaternary determinants occurred in all alpha-neoproteins. An excess of A chains did not revert alpha-neoproteins to alpha-crystallin. However, alpha-neoprotein molecules did interact with added B chains forming neomolecules with lower A to B chain ratios.

Molecular evolution and subunit structure of cattle lens alpha crystallin.

The present studies were based on the premise that any common determinants in homologous proteins must have originated with the common ancestor of all of the taxonomic groups in which that determinant occurs. Cross-reacting antigenic determinants of lens alpha crystallin various classes of modern vertebrates were used to trace their evolutionary relationships. For quantitation of evolutionarily distinct determinants, equimolar amounts of alpha crystallin or its subunits, in either monomeric or reaggregated form, were bound to a matrix, then saturated with 125I-labeled Fab fragments of anti-cattle alpha crystallin antibodies having phylogenetically restricted specificities. This quantitative procedure has the important advantage of independence from variation in antibody responses to different determinants of the same antigenic molecule. The procedure is not impaired by steric hindrance. Both the SH-containing and SH-free subunits of cattle lens alpha crystallin were found to contain common antigenic determinants with the cyclostomata alpha crystallin. Such determinants originatd in evolution with the first vertebrates, the primitive agnatha. Antigenic determinants transferred from ancestral aquatic and land vertebrates to the mammals were found to constitute 93% of all determinants reactive in the monomeric SH-free subunits of cattle alpha crystallin. These determinants constitute only 76.5% of all determinants which are reactive in the SH-containing subunits. The antigenic determinants on both types of subunits were all found to be different. These findings indicate that evolutionary changes must have occurred more slowly in SH-free subunits than in SH-containing subunits. Significant decreases or increases were found in the content of various evolutionarily distinct determinants reactive in the reaggregated subunits as compared to the ones reactive in monomeric subunits. These differences can result from the formation of new conformational antigenic determinants during aggregation as well as from the burial or exposure of other determinants after aggregation. Different amounts of evolutionarily distinct antigenic determinants were found to be reactive in the molecules dissociated into subunits than in the intact molecules one of the reasons being that the intact molecules contain phylogenetically distinct determinants which depend on the quaternary structure of the protein molecule. The data obtained indicate that the quaternary structure of cattle alpha crystallin has, to a large degree, remained unchanged since the origin of vertebrates.

Studies on competitive inflammatory sites. I. Influence of the size of sensitizing skin implants on second-set corneal graft reactions.

A dependence of second-set corneal graft reactions on the size of the sensitizing skin allografts was established in different rat donor-recipient pairs, each of which involved a response to the B2 major histocompatibility antigen. Sensitizing skin sizes larger or smaller than the optimal size were found to result in less intense reaction in subsequently placed second-set corneal allografts. The data obtained support the view that a persisting skin allograft not only sensitizes, but constitutes a competitive inflammatory site. The latter role of a first-set skin allograft may be facilitated by being both more accessible to inflammatory cells and larger than corneal grafts. The relative strength of the sensitizing and competitive effects of a first-set skin allograft apparently influences the fate of a second-set corneal allograft.

Lateral and transmembrane redistribution of tissue-specific antigens in single cells and monolayers.

Tissue-specific antigens in the membranes of corneal endothelial cells react with anti-tissue antibodies only in metabolically active monolayers and dispersed cells. After metabolic inhibition by exposure of these preparations to cold, the antigen-antibody complexes, like free antigens, undergo transmembrane redistribution leading to their internalization by the cells. This transmembrane redistribution is reversible and can be followed by using fluorescein-labeled antibodies. Reexpression of the complexes on the cell surfaces occurs after return from metabolic inhibition to metabolic activity. Dispersed corneal endothelial cells are also capable of lateral redistribution (capping) of the complexes although cells in monolayers do not share this capability. Capping in the dispersed cells occurs only at ambient temperatures and, because it results in shedding of the complexes, is irreversible. The data indicate that macromolecules in the membranes of cells organized in tissues are restricted in their movement as compared to the macromolecules of cells functioning in a dispersed state.

The relative capacity of corneal, heart, kidney and skin cells to stimulate allogeneic lymphocytes.

In stimultaneous experiments, the ability of inbred Wistar-Furth rat corneal cells to stimulate inbred Fisher rat lymphocytes in mixed culture was compared with the stimulatory capacity of the same number of Wistar-Furth skin, kidney, heart and lymphocyte cells. The tissue cells were dissociated and after inhibition by mitomycin C cultured with an equal number of allogeneic spleen lymphocytes for 5 days. In all of these mixed cell cultures, the allogeneic lymphocytic response was mainly to the major Ag-B2 histocompatibility antigen. The stimulatory effect of corneal cells was found to be the same as that of heart, kidney and skin cells. The data indicate a lack of differences between the density of histocompatibility antigens on the surfaces of these cells.

Cellular aspects of conjunctival inflammation induced by the synergistic action of histamine and prostaglandins.

Histamine or prostaglandin (PG) E1 or E2 administered to rabbits topically alone in high doses produced conjunctival vasodilation associated with little or no edema while their mixture at lower concentrations produced conjunctival vasodilation associated with profound edema. Sections of tissues treated with the mixture of histamine and PGE1 or PGE2 showed widespread epithelial and subepithelial inflammatory cellular infiltration. Conjunctival smears from eyes treated with the histamine/PG mixture contained small lymphocytes and polymorphonuclear leukocytes, including eosinophilic and occasionally basophilic cells. Differential staining of the polymorphs demonstrated both eosinophils and pseudoeosinophils. Histological examination of the conjunctival smears and sections of the lids obtained from eyes treated with either histamine or PGE1 or PGE2 alone did not show any detectable increase of inflammatory cells when compared to normal controls. The clinical and histological results indicate that the synergistic effect of histamine with PGs of the E-type in the conjunctiva produces an inflammatory response similar to that seen in various clinical forms of human allergic conjunctivitis. Such a response could not be produced by histamine or PGE1 or PGE2 alone even at much higher doses than in the mixture. The data indicate that an interplay of several different mediators may be crucial in the conjunctival response in allergy.