Marjorie Shih

Sequence analysis of βA3, βB3, and βA4 crystallins completes the identification of the major proteins in young human lens

A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as αA, αB, βA1, βA3, βA4, βB1, βB2, βB3, γS, γC, and γD-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human βA1, βA3, βA4, and βB3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of βA3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.

Mass measurements of C-terminally truncated alpha-crystallins from two-dimensional gels identify Lp82 as a major endopeptidase in rat lens.

Molecular chaperone activity of lens α-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on α-crystallins, 2) identify α-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo. Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 mm Ca2+. Resulting fragmented α-crystallins were separated by two-dimensional gel electrophoresis. Eluted α-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble α-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of αA-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo, the protease-specific truncations removing 5 and 11 residues from αA were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of αA. Using uniquely truncated αA-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of α-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications.

Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses

Purpose. To determine if proteolysis by the calcium-activated protease m-calpain (EC 34.22.17) enhances in vitro light scattering in crystallins from human and bovine lenses. Methods. Total soluble proteins from bovine, human, and rodent lenses, ßH crystallin, or recombinant ßB1 polypeptide were pre-incubated in the presence or absence of activated m-calpain. Heat-induced light scattering was assayed by measuring changes in optical density at 405 nm. Pro-teolysis and cleavage sites were detected by SDS-PAGE, two dimensional electrophoresis, and N-terminal Edman sequencing. Results. The in vitro cleavage sites produced by m-calpain on the N-termini of human ßB1, ßA3, and ßB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of a- and ß-crystallins was associated with enhanced, heat-induced light scattering by human and bovine lens proteins. Conclusions. Proteolysis may be a contributing factor in the insolubilization of crystallins occurring during normal maturation of lens or during cataract formation in such species as man and cows.

Precipitation of crystallins from young rat lens by endogenous calpain

The purpose of these experiments was to study the mechanism for precipitation of lens crystallins in cataract. An in vitro model was developed to activate the endogenous protease calpain II in the soluble proteins from young rat lens by addition of calcium in the presence of 120 mM KCl. Light-scattering, insoluble proteins were produced approximately 4-6 days after calpain II activation. Results showed that proteolysis was caused by activation of lens calpain II, proteolysis preceded precipitation by several days, and alpha-crystallin acted as a molecular chaperone against precipitation of crystallins caused by proteolysis. These data supported our hypothesis that calpain-induced proteolysis of the N-terminal arms of beta-crystallin polypeptides leads to a loss of normal oligomerization of beta-crystallin polypeptides and formation of abnormal insoluble aggregates, possibly stabilized by hydrophobic interactions.

Crystallins from rat lens are especially susceptible to calpain-induced light scattering compared to other species

PURPOSE To compare the susceptibility of crystallins from various animal species to formation of light scattering elements after proteolysis by calpain II enzyme (EC 3.4.22.17). METHODS Lens, total soluble proteins from: 12-day and 4-week old rat, fetal and adult bovine, 16-day embryonic and 10-week chicken, and young human cortex and nucleus were proteolyzed by either endogenous lens calpain or addition of purified calpain II for 24 h followed by incubation for up to 11 days. Absorbance of light at 405 nm estimated light scattering by crystallins; SDS-PAGE and 2D-electrophoresis assessed proteolysis on the crystallins. RESULTS Most rapid light scattering occurred with total soluble proteins from young rat lens, either after adding purified calpain or by activating endogenous lens calpain with calcium. (Only rat lens showed activation of endogenous calpain II.) beta-crystallin polypeptides from rat, bovine, human, and to a more limited extent, chick lens were partially proteolyzed by addition of purified calpain II. In spite of this proteolysis, total soluble proteins from chicken, bovine, and human lenses showed no obvious light scattering by action of calpain. Crystallins from older rat lens showed approximately 50% of the light scattering displayed by crystallins from younger rats after 3 days, but only when purified calpain was added. CONCLUSIONS Our results indicate an unusually high susceptibility of crystallin polypeptides from young rat lens to formation of light scattering elements after limited proteolysis. Thus, young rat lens provides a unique opportunity to investigate how properties of crystallins influence the development of light scattering found in cataract.

Hemoglobin Chico [β66(E10)Lys→Thr]: A New Variant with Decreased Oxygen Affinity

Hemoglobin Chico was discovered in an asymptomatic 3-year-old boy when a mild anemia was detected by a routine blood count. Affected individuals in three generations are also mildly anemic. The abnormal hemoglobin amounts to about 45% of the total. It separates from Hb A by cellulose acetate electrophoresis at pH 8.5 with a mobility similar to Hb J but does not separate in citrate ayar at pH 6.2. Stability in isopropanol is slightly decreased. Its structure differs from the normal by the substitution of a threonyl residue for lysyl residue at position 66(E10) of the 8 chain. The P50 of the oxygen equilibrium curve of whole blood at 37°C was 38 torr compared with controls of 27±2 torr. The P50 binding studies of the isolated Hb Chico revealed a unique right shift of the equilibrium curve with an oxygen binding constant (1/P50) about half of normal. The remaining allosteric properties were essentially normal. This significant decrease in oxygen affinity appears to be due to changes in the heme region which ...

Hemoglobin Linkoping [β36 (C2) Pro→Thr] in a Large Finnish Family from Astoria, Oregon, USA

Eleven members of a large Finnish family from Astoria, Oregon were studied because of an erythrocytosis. No abnormality was detected by the usual hemoglobin electrophoretic tests, but an abnormal variant was separated by reverse phase HPLC. All of the affected individuals have an increased oxygen affinity with a P50 for whole blood at 37 degrees C averaging 18 torr. Fifty percent of their hemoglobin was found to have a threonyl residue in place of the normal prolyl residue at position 36 (C2) of the beta globin chain. This abnormality is identical to Hb Linkoping which was recently reported in a Finnish man living in Sweden.

Expression of calpain small subunit 2 in mammalian tissues

Purpose. The purpose of the current experiments was to more closely define the distribution and the function of calpain small subunit 2 (css2). Css2 is a newly discovered regulatory protein for the calcium activated proteases, μ- and m-calpains. Methods. Tissues from rat, monkey, and man of various ages were used to determine expression patterns of css2 by relative quantitative RT-PCR using 18S rRNA as an endogenous standard. Recombinant css2 and the 80kDa catalytic subunit of m-calpain (80 kDa/css2) were co-expressed in Escherichia coli. Casein zymography was used to measure the enzymatic activity of 80 kDa/css2 proteins. Lens α-crystallin and βB1-crystallin were used as substrates to determine proteolysis by 80 kDa/css2. Computer-based homology modeling was used to predict interactions between the traditional small subunit (css1) or css2 with the 80kDa catalytic subunit. Results. Css2 appears to be a functional equivalent of css1 in vitro in that the calcium-dependent proteolytic activity of 80 kDa/css2 was similar to recombinant m-calpain (80 kDa/css1). In rat and human lens, css2 transcripts increased with age, whereas css1 transcripts decreased with age. Human βB1-crystallin and rat αA-crystallin were cleaved similarly by 80 kDa/css2 and 80 kDa/css1. Interestingly, αA-insert crystallin was not hydrolyzed when css2 was substituted for css1 in the calpain dimer, suggesting that css2 may perform different functions from css1 in terms of proteolysis of lens crystallins during maturational growth of the lens. Css2 may also assist in the proper folding of the 80kDa subunit and regulate protease activity in the absence of calcium. Conclusions. The wide distribution of css2 transcripts in rat and monkey suggested that css2 is a second, widely distributed (rather than tissue-specific) calpain small subunit, in addition to the long-recognized css1. Further studies at the protein level will indicate if css2 has unique functions apart from css1.

Calpain Isoforms in the Eye

Retina and lens from rodents contain at least five newly discovered isoforms of calpain (Lp82, Lp85, Rt88, Rt88′ and Rt90), in addition to the ubiquitous calpains. Unexpectedly, these isoforms were related to muscle-preferred p94. Although some of their biochemical characteristics are known, the current challenge is to discover the functions of calpain isoforms under normal and pathological conditions. Further, because of stop codons, orthologues of the isoforms discussed above do not exist in man. Another challenge is to relate the rodent data to the human situation. For example, do undiscovered calpain isoforms performing the same functionsas Lp82, Lp85, Rt88 and Rt90 exist in human eye?