Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry.

Characterization of proteins that mediate mechanotransduction by hair cells, the sensory cells of the inner ear, is hampered by the scarcity of these cells and their sensory organelle, the hair bundle. Mass spectrometry, with its high sensitivity and identification precision, is the ideal method for determining which proteins are present in bundles and what proteins they interact with. We describe here the isolation of mouse hair bundles, as well as preparation of bundle protein samples for mass spectrometry. We also describe protocols for data-dependent (shotgun) and parallel reaction monitoring (targeted) mass spectrometry that allow us to identify and quantify proteins of the hair bundle. These sensitive methods are particularly useful for comparing proteomes of wild-type mice and mice with deafness mutations affecting hair-bundle proteins.

Lysine acetyltransfer supports platelet function.

BACKGROUND AND OBJECTIVES: The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function. METHODS AND RESULTS: Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of the identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments showed that platelets express p300, which is activated in an Src kinase-dependent manner upon platelet stimulation with the platelet glycoprotein VI agonist collagen-related peptide (CRP). Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin, and cortactin, as well as F-actin polymerization, integrin activation, and platelet aggregation. Super-resolution visualization of platelet actin-rich adhesion structures revealed abundant acK protein colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodium formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiologic role for lysine acetyltransferase activity in platelet function. CONCLUSION: Together, our findings reveal lysine acetyltransfer to be a potential regulator of platelet actin dynamics, and potential roles for lysine acetylation in the molecular coordination of platelet activation and function.

Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-ß and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell-cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

Diagnostic and therapeutic potential of tetanus toxin-derivatives in neurological diseases.

We assessed the ex vivo reactivity of peptidic constructs of Tet1 (analog of tetanus toxin non-virulent C fragment) with sequence homology to the cysteine-active site of thioredoxin (Tet1THO) or tetralysine (Tet1PLYS) with oxidative species or axonopathic sodium cyanate (NaOCN), respectively. We then assessed their neuronal uptake in vivo in laboratory animals. The reactivity of Tet1PLYS with NaOCN (1:2.5 to 1:37.5 molar ratios) or Tet1THO with hydrogen peroxide (1:0.4 to 1:6.2 molar ratios) was assessed by mass spectrometry. Green fluorescence protein (GFP)-tagged Tet1-derivatives (3 mg/ml in artificial cerebrospinal fluid) were administered daily to rats by intramuscular injection in latissimus dorsi at lumborum at the dose of 1 µl/g of body weight, for 3 days. Motor neuron uptake was assessed after double immunolabeling for GFP and choline acetyltransferase. Mass spectrometry analysis successfully demonstrated the ex vivo reactivity of Tet1-derivatives in a concentration-dependent manner. Confocal microscopy revealed the localization of Tet1-derivatives in axons and motor neuron cell bodies. Intramuscular delivery of Tet1-derivatives appears to be a practical approach to circumvent the blood nerve barrier and selectively deliver small molecules to the nervous system, for diagnostic and/or treatment purposes.

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity.

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin-agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.

Biochemical properties of lens-specific calpain Lp85.

Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K(50%act)=20 microM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on alphaA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

Purification and characterization of lens specific calpain (Lp82) from bovine lens.

Ubiquitous type m-calpain and lens specific Lp82 calpain were separated and partially purified from fetal bovine lens and the enzymatic characteristics were compared. Lens m-calpain required 200 microM calcium for 1/2 maximal activity, while Lp82 required 30 microM. Both types of calpains were inhibited by 0.1 mM E64, and 5 mM iodoacetamide, but not by 1 mM phenylmethylsulfonyl fluoride. Lp82 was insensitive to 1 microM calpastatin peptide while m-calpain was effectively inhibited. In the presence of calcium, m-calpain lost most of its activity within 2 hr, while Lp82 was continually active for 18 hr. Both calpains cleaved the natural substrates betaA3 and alphaB crystallins in a similar manner. However, incubation of alphaA crystallin with m-calpain removed ten amino acid residues from its C-terminus, while incubation with Lp82 removed only five residues. The latter truncation product of alphaA was also found in vivo. These data suggested that Lp82 may have a more important role than m-calpain in modification of crystallins during lens maturation.

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.

Resistance of human betaB2-crystallin to in vivo modification.

Post-translational modifications and/or structural changes induced by modifications are likely causes of the decrease in crystallin solubility associated with aging and the development of cataract. Characterization of human lens crystallins by mass spectrometry has demonstrated that betaB2-crystallin undergoes less modification than any of the other crystallins. As the lens ages, betaB2-crystallin retains its hydrophilic N-terminus while the hydrophilic C-termini of alpha-crystallins and large portions of the N-termini of betaA3/A1 and betaB1 are truncated. The hydrophilic terminal regions of crystallins contribute to their solubility. Furthermore, deamidation and disulfide bond formation, other modifications that may affect solubility by altering conformation, are less extensive in betaB2 than in the other crystallins. This resistance to modification results in higher levels of betaB2 compared with the other crystallins in the water-soluble fraction of older lenses. The solubility of betaB2 and its propensity to form non-covalent associations with less soluble beta-crystallins may contribute to the solubility of the other beta-crystallins. A current hypothesis is that the chaperone-like properties of alpha-crystallins contribute to lens crystallin solubility, particularly in younger lenses. In older lenses, where most of the alpha-crystallins have become water-insoluble, betaB2-crystallins may play a dominant role in lens crystallin solubility.

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.

Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice.

PURPOSE: The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice. METHODS: Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day-old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples. RESULTS: Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of alphaA-crystallin. CONCLUSIONS: Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

Isolation and identification of Prevotella tannerae from endodontic infections.

Black-pigmented bacteria are often isolated from endodontic infections. Five strains of black-pigmented bacteria isolated from endodontic infections could not be identified in our laboratory. The purpose of this study was to sequence the 16S rRNA gene of the five unknown isolates and identify the organisms. The 16S rRNA genes from the unknown organisms were cloned, sequenced, and determined to be Prevotella tannerae. In addition, samples from endodontic infections were surveyed for the presence of the organism. When 118 samples from endodontic infections were examined using polymerase chain reaction with specific primers for P. tannerae, 60% of the samples were positive for the presence of the organism. This suggests that P. tannerae is commonly present in endodontic infections and could be a potential pathogen.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polymerase chain reaction for differentiation of Prevotella intermedia and Prevotella nigrescens.

Isolates previously thought to be Prevotella intermedia have been shown to be a closely related species now known as Prevotella nigrescens. The purpose of this study was to determine the efficacy of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) to differentiate endodontic isolates of P. nigrescens from P. intermedia. Fifty-six strains of black-pigmented bacteria isolated from endodontic infections and conventionally identified as P. intermedia were used in this study. Using SDS-PAGE, novel polypeptide bands were used to differentiate P. nigrescens from P. intermedia. PCR was accomplished with specific primers for the 16S ribosomal RNA gene of both strains. Of 56 endodontic isolates, 41 (73%) strains were identified by SDS-PAGE as P. nigrescens and 15 (27%) strains as P. intermedia. Of the 41 strains of P. nigrescens identified by SDS-PAGE, PCR identified 37 strains as P. nigrescens. Restriction endonuclease digestion of amplified 16S ribosomal RNA genes indicated that the remaining four strains originally identified by SDS-PAGE as P. nigrescens were actually strains of Prevotella distinct from P. nigrescens and P. intermedia. Of 15 strains of P. intermedia identified by SDS-PAGE, PCR identified 14 strains as P. intermedia; but, one strain was identified as P. nigrescens. The results indicated that PCR was a more precise method than SDS-PAGE to differentiate P. intermedia from P. nigrescens. This study confirms that P. nigrescens is more commonly isolated in pure culture from endodontic infections than P. intermedia.

Lens cytoskeleton and transparency: a model.

The function of the cytoskeleton in lens was first considered when cytoplasmic microtubules were observed in elongating fibre cells of the chick lens nearly 40 years ago. Since that time, tubulin, actin, vimentin and intermediate filaments have been identified and found to function in mitosis, motility and cellular morphology during lens cell differentiation. A role for the cytoskeleton in accommodation has been proposed and modification of the cytoskeletal proteins has been observed in several cataract models. Recently, a progressive increase in protein aggregation and lens opacification was found to correspond with the loss of cytoskeletal protein in the selenite model for cataract. In the present report a model is proposed for the role of tubulin, actin, vimentin, spectrin and the lens-specific filaments, filensin and CP49, in the establishment and maintenance of transparent lens cell structure.

Survey for collagenase gene prtC in Porphyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections.

Collagenase is a potential virulence factor shown to be expressed by Porphyromonas gingivalis associated with periodontal disease. The purpose of this study was to use the polymerase chain reaction (PCR) to detect the presence of the collagenase gene (prtC) in 21 strains of Porphyromonas species isolated from endodontic infections. Type strains for P. gingivalis (ATCC 33277), P. endodontalis (ATCC 35406), Prevotella intermedia (ATCC 25611), and Prevotella nigrescens (ATCC 33563) were used as controls. When PCR primers specific for the 16S ribosomal RNA gene of P. gingivalis or P. endodontalis were used, 16 of the strains were identified as P. gingivalis, and five strains were identified as P. endodontalis. The presence of the prtC gene for collagenase was detected using PCR. Amplicons were analyzed by agarose gel electrophoresis, with an 815 bp amplicon representing the presence of the collagenase gene. Type strain ATCC 33277 and all 16 clinical isolates of P. gingivalis produced the collagenase gene amplicon. Neither type strain ATCC 35406 nor the five strains from clinical isolates of P. endodontalis produced the collagenase gene amplicon. These results indicate that P. gingivalis from endodontic infections possesses the prtC gene. P. endodontalis does not seem to exhibit prtC. The virulence of P. gingivalis may be related to its production of collagenase.

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.

Local microdomain structure in the terminal extensions of betaA3- and betaB2-crystallins.

PURPOSE: Although the crystal structures of the core domains of bovine betaB2-crystallin have been determined and those of other betagamma-crystallins modeled, the positions of the N- and C-termini are not resolvable by X-ray crystallography. Here we model the possible structural organization of the terminal arms of mouse betaA3- and betaB2-crystallins and test this model against the results of partial proteolysis. METHODS: The secondary structure of the terminal extensions was predicted by 3 different methods, one a nearest-neighbor method modified to use overlapping sequence tripeptides. Recombinant betaA3- and betaB2-crystallins were expressed using baculovirus vectors in S. frugiperda Sf9 cells. Crystallins were sequenced by the Edman degradation method. RESULTS: The N-terminal extension of betaB2-crystallin includes a series of hydrophilic residues from Q-11 to Q-9 which have high propensity of a helical conformation. The N-terminal arm of betaA3-crystallin is also predicted to have two helical segments, from Q-24 to E-20 and M-13 to A-12. Partial characterization of the baculovirus extract showed a thiol protease inhibited by leupeptin and E-64. As predicted by the model, recombinant betaB2-crystallin subjected to partial proteolysis was cleaved adjacent to the helical domain, while the N-terminal cleavage site in recombinant betaA3-crystallin was within 1 residue of an interhelical junction. Our model also predicts the products of partial proteolytic degradation of betaB2- and betaA3-crystallins from human, rat, bovine and chicken lenses incubated with the protease m-calpain. CONCLUSIONS: These results suggest the existence of local microdomain structures in the N- and C-terminal extensions of betaA3- and betaB2-crystallins, which appear to be more susceptible to proteolytic degradation in regions adjacent to these putative domains.

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.