Grazyna Adamus

Anti-rhodopsin monoclonal antibodies of defined specificity: Characterization and application

A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbent assay (ELISA) in the presence of synthetic peptides from rhodopsins cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsins amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 118-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 10(8)-10(10) M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsins carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similarly, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

Binding pattern of anti-rhodopsin monoclonal antibodies to photoreceptor cells: An immunocytochemical study

A panel of anti-rhodopsin monoclonal antibodies (MAbs) of defined epitope specificity has been evaluated by immunocytochemistry. Most of the IgG class MAbs (23/27) gave positive results, but only a few of the IgM class MAbs (2/21) were useful for this application. MAbs specific to the N-terminal region stained rod outer segments most strongly, with progressively less staining in the Golgi, perikarya, plasma membrane of the inner segment, and synaptic region. Phagosomes located basally in the pigment epithelium were stained; cone cells were consistently negative. Antibodies to the C-terminus of rhodopsin labeled the same cell structures (except for phagosomes) but showed diversity in their binding pattern. Many of these MAbs bound to cone outer segments in addition to rods, and showed different patterns of binding to red/green and blue cones. Antibodies specific for rhodopsin sequence 340-348 labeled different types of cone cells, indicating differences in their binding sites. Two MAbs were found to label hydrophilic loop sequences which connect rhodopsins transmembrane segment: MAb K42-41 which binds loop 5-6, and MAb A1-55 which binds loop 2-3. At least these two regions of the rhodopsin sequence in addition to the C- and N-termini, are available for antibody reaction in fixed retina.

Early expression and localization of rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) in the developing fetal bovine retina

Differentiation and maturation of the photoreceptor outer segments are key steps in the development of the visual system. Morphological studies presented here show that the cow and human are nearly identical in the timing of outer segment appearance during fetal development, implying that the bovine retina is a good model system for the final stages of human photoreceptor development. To study photoreceptor maturation, rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) were quantified by ELISA in a developmentally staged series of fetal bovine retinas. In addition, their localization within these retinas was determined by immunogold electron microscopy. Rhodopsin, as detected by antibodies directed against either the N- or C-terminal portions of the molecule, is first found at about 5.5 months gestation. It is first detected on the plasma membrane of the immature cilia and on the earliest emergent outer segment membrane, even before organized disk membranes are apparent. In contrast, whereas rhodopsin levels and outer segments are nearly undetectable before 5 months gestation, IRBP accumulates to a significant level (4-5% of the adult) as early as 3 months gestation. Immunogold electron microscopy confirmed this finding, with localization of IRBP predominantly in the subretinal space.

Induction of experimental autoimmune uveitis with rhodopsin synthetic peptides in Lewis rats

Rhodopsin, a membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) in Lewis rats. Synthetic peptides derived from rhodopsin sequences that cover hydrophilic, exposed regions of the protein were tested for their capacity of eliciting in vitro T cell proliferation and their ability for inducing EAU in Lewis rats. Rats were injected with rhodopsins peptides mixed in complete Freunds adjuvant containing M. tuberculosis H37Ra (5 mg/ml) three days after pretreatment with cyclophosphamide (20 mg/kg). ELISA results indicate that all peptides induce antibody responses; however antibody titers differ among sera tested. Immunization with four peptides--the amino-terminus (2-32), loop I-II (61-75), loop V-VI (230-251), and the carboxyl-terminus (324-348 and 331-342) induced both antibody and T cell responses. In all cases, the proliferative responses of cells derived from peptide-injected rats were stronger against the immunizing peptide than against native protein. Three distinct uveitogenic epitopes were identified on rhodopsins cytoplasmic surface--within the rhodopsin carboxyl-terminus (324-348), loop I-II (61-75), and loop V-VI (230-250). Histopathologically, at the immunized doses, total destruction of the photoreceptor cell layer was observed as compared to the control group. Loop V-VI caused severe inflammation of the retina while the other pathogenic peptides produced less severe destruction with few inflammatory cells present. Our study indicates that the major immunodominant T cell epitope (331-342) is also involved in EAU induction but is not the primary uveitogenic site.

Genetic control of antibody response to bovine rhodopsin in mice: epitope mapping of rhodopsin structure

Inbred strains of mice of independent haplotype were immunized with bovine rhodopsin. All mice tested except SJL developed significant titers of specific antibodies 21 days after a single immunization. Anti-rhodopsin antibody level differed among conventional inbred strains. Comparison of the immune response to rhodopsin of congenic mice on two different genetic backgrounds showed that animals with an A background typically produced higher levels of specific antibody than mice with a B10 background. Titer of specific antibodies in antisera of mice of the same H-2 haplotype but different Igh haplotype differed; e.g. for H-2d haplotype, NZB (Ighn) generated the highest level of antibody with BALB/c (Igha), DBA/2 (Ighc), and B10.D2 (Ighb) strains giving successively lower responses. The location of immunodominant regions of bovine rhodopsin was investigated in primary sera among strains of mice. Sera were tested for their binding of anti-rhodopsin antibodies to synthetic peptides covering the entire primary structure of rhodopsin. From direct binding studies with hydrophilic rhodopsin peptides, the majority of the antigenic binding sites were localized in the sequence of the amino terminus, the II-III loop and the carboxyl terminus. Binding to these antigenic peptides was not strain restricted. Application of the overlapping synthetic peptide strategy of Geysen enabled refinement of these epitopes and determination of an additional major epitope in the hydrophobic sequence 304-310.

Molecular, enzymatic and functional properties of rhodopsin kinase from rat pineal gland

Rhodopsin kinase activity from rat pineal gland and from rat retina are indistinguishable, based upon determination of a variety of enzymatic and molecular properties. Both activities are independent of calcium, cyclic nucleotides, and calmodulin. Both are activated by spermine and inhibited by adenosine and some rhodopsin kinase specific adenosine derivatives such as sangivamycin. The Kms for rhodopsin, ATP, and GTP are indistinguishable for the protein kinase in extracts from the retina and from the pineal gland. The apparent molecular weight of the kinase from both sources, as determined by gel filtration and autoradiography of the 32P-labeled autophosphorylated kinase, is about 70 kDa. Rhodopsin kinase activity from pineal binds in a light-dependent manner to rhodopsin in rod outer segments as does the enzyme from retina. Monoclonal antibodies against bovine rhodopsin were used in an immunochemical study that identified a rhodopsin-immunoreactive protein in rat pineal gland and retina. Using an ELISA we demonstrated the presence of a rhodopsin-immunoreactive protein in rat pineal gland equivalent to 0.075 pmol rhodopsin per gland. Frog pineal organ (Rana catesbiana) contains 33 times more of this rhodopsin-like protein than does rat pineal gland.

Alligator rhodopsin: Sequence and biochemical properties

We sequenced selected peptides of alligator rhodopsin that accounted for about half of the total protein. These sequences were confirmed when the total primary structure of alligator rhodopsin was deduced from the cDNA sequence. Differences in the amino-terminal region, compared to that of bovine rhodopsin, account for failure of cross-reactivity of several anti-bovine rhodopsin monoclonal antibodies. Differences in the carboxyl-terminal region give rise to limited antibody cross-reactivity and may also account for a slightly reduced ability of alligator rhodopsin to be phosphorylated by bovine rhodopsin kinase. Alligator rhodopsin regenerates much faster than bovine rhodopsin. The pseudo-first-order rate constant for alligator rhodopsin regeneration is approximately 25 times that of bovine. Phylogenetic analysis of 17 rhodopsin sequences indicates that the alligator is more closely related to the chicken than to the other species examined.

Specificity of T and B cell responses to bovine rhodopsin in Lewis rats

Rhodopsin, an integral membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) when injected into Lewis rats. This disease is characterized by a mononuclear and polymorphonuclear cellular infiltrate of the retina resulting in destruction of the photoreceptor cells. In this study the B and T cell specificities of the response to bovine rhodopsin by Lewis rats were determined. Antibodies induced by injection of rhodopsin were directed almost exclusively to the IV-V loop (residues 174-202). Later in the response, antibody to the N-terminus was also detected. At the T cell level, most activity was directed to the C-terminus as measured by in vitro lymphocyte proliferation. Other minor T cell epitopes were found in the II-III (96-114) and IV-V (174-202) loops. Further dissection of the amino acid sequence responsible for the activity isolated to the C-terminus indicated that a 12-amino acid-long sequence (331-342) elicited the strongest proliferative response.

A practical method for rescuing desired hybridomas during monoclonal antibody production

SummaryWe present a practical method for the rescue of previosly stable hybridoma clones which increases the proportion of desired cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting to isolate a hydridoma cell line that is relatively rare in a population.

Peptides from Rhodopsin Induce Experimental Autoimmune Uveoretinitis in Lewis Rats

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T cell mediated autoimmune disease, which is characterized by destruction of the photoreceptor cells of the retina. EAU serves as a model for human intraocular inflammatory diseases, like uveitis, which are major causes of visual impairment. The animal disease can be induced by immunization with certain retinal proteins. The antigens used in most studies for EAU induction have been S-antigen (arrestin) and interphotoreceptor retinoid binding protein (IRBP)(Gery, Michizuki and Nussenblatt, 1986). We, and other investigators, have reported that rhodopsin is similarly uveitogenic in rats, guinea pigs, and in monkeys (Adamus, et al., 1992; Marak, et al., 1980; Meyers-Elliott and Sumner, 1982; Moticka and Adamus, 1991; Schalken, et al., 1988b; Schalken, et al., 1989; Wong, et. al., 1977). Rhodopsin is the major protein of photoreceptor cells and has a molecular weight of 40,000 daltons. It is the photoreceptor protein that initiates the visual transduction process (Hargrave and McDowell, 1993).