Martin J. McDermott

Phosphorylation of α-crystallin B in Alexander's disease brain

The phosphorylation of α‐crystallin B was studied in homogenates of autopsy samples of brain tissue from patients with Alexanders disease, a condition characterized by over‐expression of this protein. After incubation in the presence of [γ‐32P]ATP and cAMP the homogenates were analyzed by two‐dimensional electrophoresis, (isoelectric focusing followed by SDS‐PAGE). Three major polypeptides having the same molecular weight as bovine lens α‐crystallin B and pIs 7.1, 6.9 and 6.7 were detected in the Coomassie blue stained gels. These three polypeptides were recognized by an α‐crystallin B‐specific antiserum in Western blots. The polypeptides with pIs 7.1 and 6.7 co‐migrated in isoelectric focusing gels with bovine lens αB and its phosphorylated form αBp, respectively. Radioautography of the two‐dimensional gels demonstrated the presence of 32P in the most acidic polypeptide. The results demonstrate the occurrence of αB phosphorylation in Alexanders disease brain tissue.

Differential synthesis and phosphorylation of the α-crystallin A and B chains during bovine lens fiber cell differentiation

[14C]-amino acids and [32P]-orthophosphate incorporation experiments were carried out in bovine lenses in culture to study the synthesis and phosphorylation of alpha-crystallin A and B polypeptides during differentiation of the lens fiber cells. Following culture, the [14C] or [32P]-labelled alpha-crystallin was isolated by gel filtration chromatography from four regions of the lens corresponding to: A) quiescent epithelial cells, B) dividing epithelial cells and early stages of elongation, C) young elongating fibers, and D) mature fibers from the superficial cortex. The incorporation of label into the alpha-crystallin primary gene products alpha A2 and alpha B2 and their respective phosphorylated forms alpha A1 and alpha B1 was determined by isoelectric focusing and radioautography. Different synthesis and phosphorylation patterns were observed in alpha A and alpha B polypeptides. Synthesis and phosphorylation of the alpha B chain occurs most actively in the epithelial cells, both processes decrease during differentiation and there is no net accumulation of the phosphorylated form alpha B1 in the mature fiber cell. In contrast to the B chain, the A chain synthesis, minimal in the epithelial cell, increases with differentiation. Most striking, the A chain phosphorylation, not detectable in the epithelial cells, increases with differentiation. In the mature fiber cell, the phosphorylated form alpha A1 accounts for one third of the A chain. These observations indicate that the two chains may have different functions. the synthesis and phosphorylation patterns of alpha A suggest a lens-specific function of this polypeptide in the fiber cell and in the terminal differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)

The apparent molecular size of native α-crystallin B in non-lenticular tissues

The apparent molecular size of the native α‐crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an α‐crystallin specific antiserum. Native α‐crystallin from cytosol preparations of rat lens cortex was used as a reference. α‐Crystallin B present in all three cytosol preparations from non‐lenticular tissues eluted in a single symmetrical peak, with the same elution volume as α‐crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 × 106 Da. No other species could be detected. The results indicate that the α‐crystallin aggregates characterized by an apparent average molecular mass of 0.8 × 106 Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these α‐crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of αA and αB polypeptides and their respective phosphorylated forms αAp and αBp, are similar in size to those found in heart, brain and retina, containing the αB but not the αA polypeptide.

MOLECULAR CHANGES DURING THE PHOTOOXIDATION OF α-CRYSTALLIN IN THE PRESENCE OF UROPORPHYRIN

Singlet oxygen reacts preferentially with three amino acids in proteins, His, Trp and Met. In order to study the specific molecular events that result from such oxidations, calf a‐crystallin was photooxidized in the presence of uroporphyrin and the reactions were investigated by high performance liquid chromatography peptide mapping using a photodiode array detector followed by fast atom bombardment mass spectrometry (FAB‐MS). From these studies, the following conclusions can be inferred: (1) Upon photooxidation residue Met‐68 of the B chain is oxidized to Met sulfoxide, whereas residue Trp‐60 remains intact. (2) Two of the 16 His residues in a‐crystallin are photooxidized with an apparent pKa of ca 7.0. (3) FAB‐MS analysis suggests that residue Lys‐166 close to the C‐terminal end of the A chain forms a cross‐link with the His‐7 residue close to the N‐terminal end of the A chain. This may be either an inter‐ or intramolecular cross‐link.

Raman spectroscopic evidence for a disulfide bridge in calf γII crystallin

Abstract Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical γII crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500–2600 cm−1), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of γII as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500–540 cm−1), the native form shows a line near 511 cm−1 which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:1 computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm−1. In every other spectral region, both native and reduced γII forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution.

The disulfide content of calf γ-crystallin

Abstract The disulfide content of calf γ-crystallin polypeptides has been investigated. The γ-crystallin fraction of the soluble lens proteins was separated into five distinct polypeptides and characterized by isoelectric focusing, amino acid composition, and N-terminal sequence analysis to 25 residues. It has been demonstrated that 7 cysteines are present in γII,4 to 5 cysteines in γIIIa, γIIIb, and γiv, and 6 cysteines in γI (β s ). Reduction of the total γ-crystallin fraction with DTT resulted in an increase of approximately 1 to 1.5 mol of free SH per mole of protein. This increase in sulfhydryls was demonstrated to be contributed primarily by γII, the major polypeptide representing 50% of the total γ-crystallin, which showed an increase of approximately 2.5 mol of sulfhydryl per mole of protein upon reduction. Insignificant disulfide content was present in γIII and γIV and only a slight amount of disulfide was found in γI (β s ). The observed increase in sulfhydryl content upon reduction was not due to the presence of mixed disulfides of 2-mercaptoethanol, glutathione, or cysteine. The data are consistent with approximately 1 mol of intramolecular disulfide per mole of protein being present in γII. X-ray crystallography of γII has shown that the spatial location of Cys 18 and Cys 22 in the tertiary structure permits disulfide bond formation. Sequence analysis of the four major polypeptides of γ-crystallin, γII, γIIIa, γIIIb, and γIV indicates that only γII has both Cys 18 and Cys 22 . Cys 18 is present in γIIIa, γIIIb, and γIV but Cys 22 is replaced by His 22 . It is probable that the lack of disulfide in γIIIa, γIIIb, and γIV is due to the absence of Cys 22 .

Fe2+ oxidation of α-crystallin produces a 43,000 Da aggregate composed of A and B chains cross-linked by non-reducible covalent bonds

The structure of a 43,000 Da aggregate generated from bovine lens alpha-crystallin polypeptides of 20,000 Da using Fe2+ catalyzed oxidation, was studied by sequence analysis of a 30,000 Da proteolytic fragment. Three polypeptide components were simultaneously sequenced in the electroblotted 30,000 Da fragment, corresponding to Phe114-Ser130... of the alpha A chain, and His 111-Ser135... and Ser 35-Leu49... of the alpha B chain. The relative proportions of the components suggests that the three polypeptides are present in equimolar amounts. It is concluded that the 30,000 Da fragment and, therefore, the 43,000 Da aggregate is constructed of both the A and B polypeptide chains covalently cross-linked with non-reducible bonds. At least one of these cross-links is present towards the carboxy-terminus of the A and B chains after Phe114 and His111, respectively.

The calf gamma crystallins-A Raman spectroscopic study

The solution structures of the four major components of bovine lens gamma-crystallin, gamma s, gamma II, gamma III and gamma IV are compared using Raman spectroscopy. The spectral region sensitive to the vibrational frequencies of aromatic and sulfur containing residues and to the backbone skeletal stretching modes (500-1000 cm-1), and that reflecting secondary structure (1,000-1,700 cm-1) are strikingly similar in all four gamma-crystallin fractions. These similarities are indicative of the dominant anti-parallel beta sheet structure common to all the gamma-crystallins. A comparison of the ratios of the Raman intensities at 850 cm-1 and 830 cm-1 (I850/I830), an empirical measure of the degree of hydrogen bonding of phenolic hydroxyl groups, suggests that the tyrosine residues in all the gamma-crystallin fractions are moderately hydrogen bonded. Distinct differences in the solution structures of the gamma-crystallins were observed in the higher energy end of the vibrational Raman spectra. The sulfhydryl stretching frequencies for the gamma-crystallins exhibit complex splitting patterns in the 2,500-2,600 cm-1 region. These patterns are due to the competing effects of hydrogen bonding and S-pi interactions with neighboring aromatic residues. All five proteins exhibit multiple, but distinct, thiol frequencies, suggesting that the microenvironments of the cysteine residues in these proteins are significantly different.

Purification of a 43 000 Dalton aggregate generated from α-crystallin

A procedure is presented for the purification of an aggregate of covalently linked polypeptides of α-crystallin. Using either iron catalyzed oxidation or UV irradiation, 6-12% of calf lens α-crystallin can be converted to a 43,000 Da aggregate containing non-reducible cross-linked polypeptides as indicated by SDS-PAGE under reducing conditions. The 43,000 Da aggregate generated by Fe2+ oxidation can be isolated to approximately 85% homogeneity with respect to its relative molecular weight on SDS-PAGE. The procedure can be performed in two steps; 1) gel filtration of the oxidized α-crystallin under deaggregating and reducing conditions, and 2) preparative SDS-PAGE of the 43,000 Da aggregate-enriched peak obtained by filtration. The 43,000 Da aggregate band can be recovered from the polyacrylamide gels using a new preparative electroelution technique. 1.0 ± 0.3 mg of purified 43,000 Da aggregate can be obtained from 100 mg of Fe2+ oxidized α-crystallin. The described methodology will facilitate further char...