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, betaH crystallin, or recombinant betaB1 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. Proteolysis 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 betaB1, betaA3, and betaB2-crystallins were similar to some of those on bovine and rat crystallins. Proteolysis of alpha- and beta-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.

Deamidation of human beta B1 alters the elongated structure of the dimer.

The purpose of these experiments was to determine if truncation and deamidation alter the structure of a human lens protein, beta B1-crystallin. Recombinant wild type and a deamidated form of recombinant beta B1 were expressed in Escherichia coli. Wild type beta B1 was also enzymatically cleaved to generate a physiologically-relevant truncated beta B1. Purity and size of the expressed proteins were confirmed by SDS-PAGE and electrospray ionization mass spectrometry. Size exclusion chromatography and light scattering were used to determine aggregation states of beta B1. Protein conformations were predicted from sedimentation velocity analysis. Molecular weights of 49,000 and 54,000 Da were obtained for wild type beta B1 by sedimentation equilibrium and light scattering, respectively. A sedimentation coefficient of 2.7 S was determined for wild type beta B1. Molecular weights of 54,000 and 60,000 Da were determined for deamidated beta B1 by sedimentation equilibrium and light scattering, respectively. However, deamidated beta B1 eluted earlier than wild type beta B1 on size exclusion chromatography, with an estimated molecular weight between 78,000 and 116,000 Da. Loss of the extensions of beta B1 caused abnormal association of the protein with the stationary phase during size exclusion chromatography. Wild type beta B1 was predicted to form a dimer with an elongated structure. The earlier elution of the deamidated beta B1 dimer on size exclusion chromatography suggested the dimer was less compact. Truncation caused abnormal column interactions suggesting an altered conformation. These changes are important because truncation and deamidation occur extensively in aging human lenses and may be important for senile cataract formation.

Importance of nutrition for optimum health of the periodontium.

As dental professionals become increasingly aware of the association between systemic and oral health, the importance of addressing systemic nutrition issues takes on a new urgency. Nutrition is one of the modifiable factors that impact the host's immune response and the integrity of the hard and soft tissues of the oral cavity. While nutrient deficiencies are rare in the general population of the United States, there are patient populations at high risk for nutritional inadequacies. Many of these medically compromised people are seen in dental practices on a daily basis. This article will also offer some basic recommendations for nutrition counseling.

Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.

Analysis of water-soluble crystallins from human lenses, ages 32 week fetal to 55 years has led to identification of the major modifications of the proteins comprising the lens. These modifications were identified by the masses of the proteins determined by electrospray ionization mass spectrometry after the proteins were separated by gel filtration and reversed phase high performance liquid chromatography. Examination of all the proteins isolated from the water soluble portion demonstrated that the major age-related modifications causing significant alteration in the molecular weights of the lens crystallins include truncation of the N-termini of beta B1, beta A3 and beta A1, and partial phosphorylation and C-terminal degradation of alpha-crystallins. N-terminal degradation of beta B1, beta A3 and beta A1 was evident in human lenses less than one year old, and the proportion of these truncated proteins became greater with age. Phosphorylation of alpha A- and alpha B-crystallins increased from the fetal to the 3 year old lens, but did not change with further aging. Minor components indicating truncation of the C-termini of alpha-crystallins were found in older lenses. In contrast to beta B1, beta A3 and beta A1, the masses of the major species of alpha A, alpha B, beta B2, beta A4, gamma S, gamma C, and gamma D did not change with aging. This suggested that the major modifications to these crystallins are limited to deamidation and possibly intra-molecular disulfide bonds. These data, in conjunction with the data in the accompanying manuscript, established deamidation as a common modification, since deamidation, which causes only a one dalton change in mass, is the only modification that is consistent with the absence of a detectable change in molecular weight and the observed increased acidity demonstrated in the two-dimensional gels of the accompanying paper. Other age related changes included a decrease in beta B3 (M(r) 24224), a major component of the fetal lens, which was not detected in lenses older than 3 years, and increases in the ratios of alpha B:alpha A and gamma S:gamma C.

Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry.

The purpose of this study was to identify the major protein components in adult human lenses and to analyse the specific age-related changes in these proteins using two-dimensional electrophoresis, Edman sequencing, and in conjunction with the data in the accompanying manuscript, mass spectrometry. The majority of changes in the two-dimensional electrophoretic pattern of lens proteins occurred prior to 17 years of age, and included a decrease in proteins migrating to the original positions of beta B1, beta B3, beta A3, gamma C and gamma D, and the appearance of many new species with apparent molecular weights on two-dimensional electrophoretic gels similar to beta B2 and gamma S, but having more acidic pIs. These proteins were identified as deamidated forms of beta B1 and beta A3/A1 missing portions of their N-terminal extensions. With the exception of alpha B, deamidation was detected in all crystallin species. These data indicated that a major fraction of the water-soluble protein of the adult human lens is composed of truncated beta B1 and beta A3/A1 crystallins, and that nearly all human crystallins, including the, beta-crystallins, are susceptible to deamidation. The results also provided the most detailed map to date of the identities of protein species on two-dimensional electrophoresis gels of adult human lenses.

Cleavage of beta crystallins during maturation of bovine lens.

PURPOSE: (1) Identify major crystallin proteins in fetal and adult bovine lens, (2) examine the N-termini of beta-crystallins for truncation, and (3) determine if the protease m-calpain (EC 3.4.22.17) is responsible for the cleavage of bovine beta-crystallins during maturation. METHODS: Crystallins from fetal and adult bovine lenses were analyzed by one and two-dimensional electrophoresis and Edman sequencing of separated proteins and their tryptic fragments. Identical techniques were used to analyze crystallins following their incubation with purified m-calpain. RESULTS: The identities of the major crystallins and several additional crystallin species missing portions of their N-terminal extensions were identified in the fetal bovine lens. Besides the previously identified form of betaB1 missing 15 residues from its N-terminus, forms of betaA3 >missing 11 and 22 residues were identified. With aging, the betaA3 (-22) species became a major protein in the adult bovine lens, and minor forms of betaB2 and betaB3 missing 8 and 22 residues from their N-termini, respectively, appeared. Purified m-calpain cleaved within the N-terminal extensions of bovine beta-crystallins and removed: 12 or 15 residues from betaB1; 8 residues from betaB2; 5 or 10 residues from betaB3; and 11 or 17 residues from betaA3. CONCLUSIONS: Based on the cleavage sites in vitro, m-calpain may be partially responsible for cleavage of bovine betaB1, betaB2, and betaA3 during lens maturation. However, the preference of m-calpain to remove 12 residues from betaB1, and 11 and 17 residues from betaA3, suggested that the betaB1 (-15) and betaA3 (-22) species found in vivo were produced by a different protease. This unidentified protease may have a preference for the asparagine-proline-X-proline sequence found in the N-terminal extensions of betaB1 and betaA3.

Sequence analysis of betaA3, betaB3, and betaA4 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 alphaA, alphaB, betaA1, betaA3, betaA4, betaB1, betaB2, betaB3, gammaS, gammaC, and gammaD-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human betaA1, betaA3, betaA4, and betaB3-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 betaA3/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.

The sequence of human betaB1-crystallin cDNA allows mass spectrometric detection of betaB1 protein missing portions of its N-terminal extension.

The sequence of human betaB1-crystallin cDNA encoded a protein of 251 amino acids in length. Mass spectrometric analysis of intact betaB1 from young human lens confirmed the deduced amino acid sequence. Lenses of human donors newborn to 27 years of age also contained partially degraded forms of betaB1 missing 15, 33, 34, 35, 36, 39, 40, and 41 amino acid residues from their N-terminal extensions. The similarity of the cleavage site between residues 15 and 16 in human betaB1 to the cleavage occurring in bovine betaB1 suggested that lenses of both species may contain a similar proteolytic activity. The remaining cleavage sites occurring in human betaB1 did not closely match those occurring in other species, possibly due to the widely divergent amino acid sequence of the N-terminal extension of betaB1 amoung species. Results from animal models suggest that cleavage of the N-terminal extension of betaB1-crystallin could enhance protein insolubilization and cataract in lens. However, the presence of partially degraded betaB1-crystallins in both water-soluble and water-insoluble fractions of lenses of young donors suggested that the rate that proteolyzed betaB1-crystallins become water-insoluble is relatively slow in humans.

Comparison of cell-permeable calpain inhibitors and E64 in reduction of cataract in cultured rat lenses.

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Association of calpain with insoluble pellet of rat lens.

Calpains are calcium-activated neutral proteases found in many tissues including the lens. The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10% was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30% of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates; and (2) it may activate calpain, since membrane phospholipids lower the protease's calcium requirement.

Calpain II in human lens.

The purposes of the current study were to: determine if human lenses contain calpain II (EC.34.22.17) activity, measure the effect of aging and anatomical location on lens calpain II activity, and determine if human lenses contain the endogenous calpain inhibitor calpastatin. Both enzymatic and immunologic assays indicated that human lenses contained calpain II activity. Calpain II activity was highest in the cortex of lenses from young donors, and lowest in the nucleus of aged lenses, where it was sometimes nondetectable. In some cases, calpain II activity persisted in the nucleus of lenses from donors greater than 70 years of age. Human lenses also contained endogenous calpain inhibitor (calpastatin) in excess over calpain enzymatic activity. Calpastatin activity did not decrease during aging. Although human lenses contained approximately 3% of the calpain activity found in rat lenses, calpain II may still be a major endopeptidase in human lenses. Demonstration of calpain II in human lenses suggested that calpain II could be involved in both lens maturation and cataract formation.