Raúl Chiesa

The phosphorylation sites of the B2 chain of bovine α-crystallin

Abstract The B 2 chain of bovine lens α-crystallin is phosphorylated in a cAMP-dependent reaction. By analysis of 32 P-labelled chymotryptic peptides isolated from α-crystallin obtained from lenses labelled in organ culture, two phosphorylated B 2 chain fragments were found. Sequence analysis of the fragments gave the following results: Arg-Ala-Pro-Ser-Trp-Ile-Asp-Thr-Gly-Leu and Ser-Leu-Ser-Pro-Phe corresponding to residues 56 to 65 and 43 to 47, respectively. It is established by this work that B 1 is a phosphorylated post-translational product of B 2 . Both the A 2 and B 2 chains of α-crystallin are phosphorylated at a similar site with the sequence Arg-(X)-Pro-Ser. This is an unusual site for cAMP-phosphorylation since the phosphorylated serine is preceded by a proline residue. It may also be of significance that the other B 2 chain phosphorylation site even more radically differs from previously reported cAMP-dependent phosphorylation sites.

Definition and comparison of the phosphorylation sites of the A and B chains of bovine α-crystallin

The major phosphorylation sites of bovine alpha-crystallin Ser122 in the A chain, Ser59 and Ser43 and/or Ser45 in the B chain have been previously characterized. Further analysis of total alpha-crystallin, isolated from the cortex of calf lenses incubated in the presence of [32P]orthophosphate, demonstrated the presence of additional phosphorylation sites in both chains. At least three additional phosphorylation sites were found in the A chain and at least one in the B chain. These additional sites accounted for approximately 25% of the radioactivity incorporated in the protein. Two general sequences were found in most phosphorylation sites of both chains of alpha-crystallin: (Arg/Lys)-(X)-Pro-Ser and Ser-(X)-Ser-Leu-Ser. In spite of the 57% homology in the sequences of the A and B chains, the phosphorylation sites are located, in the A polypeptide, at the C-terminal third and in the B polypeptide, at the N-terminal third. The alignment of the regions containing the phosphorylation sites of both chains (C-terminal third of the A and N-terminal third of the B chain) revealed an unexpected similarity in the relative positions of the sites in each chain.

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.

The dephosphorylation of lens α-crystallin a chain

The present communication reports the presence of a phosphoprotein phosphatase activity in bovine lens preparations which dephosphorylates αAp, the phosphorylated form of αA, one of the α-crystallin polypeptides, in a Ca2+/calmodulin dependent manner. The activity was found in soluble preparations from epithelial cells but it could not be detected in similar preparations from fiber cells. A 60,000 Mr calmodulin binding polypeptide and a 15,000 Mr polypeptide found in the epithelial cell preparations comigrated in SDS-PAGE with the A and B subunits of bovine brain calcineurin (phosphoprotein phosphatase 2B) respectively. The 15,000 Mr was specifically recognized by an anti-bovine brain calcineurin antiserum. Bovine brain calcineurin was as effective in dephosphorylating αAp as the lens preparations. Thus, it is likely that the activity present in the lens is related to this enzyme. The results indicate that the lens specific polypeptide αA may be subject to metabolic control through phosphorylation and dephosphorylation pathways regulated by cAMP and calcium, respectively. Changes in the activities of these pathways appear to occur during differentiation of the lens epithelial cell and may be related to gene regulation during the differentiation process.

Lens calcium activated proteinase: Degradation of vimentin

The lens has been shown to contain a Ca+2 activated proteinase specific for vimentin. The proteinase is present in the soluble fraction of the cortex but not in the epithelium. It is suggested that this proteinase is expressed during terminal differentiation of the epithelial cells and may be responsible for degradation of the intermediate filaments in the fiber cells. The proteinase is inhibited by EGTA but not by several proteinase inhibitors.

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.

Identification of the specific phosphorylated serine in the bovine alpha crystallin A1 chain.

Previous work (1,2,3) has indicated that the in vivo post-translational modification of the alpha crystallin primary gene product A2 is due to a specific phosphorylation process involving a serine residue located in a chymotryptic fragment with the sequence ARG-LEU-PRO-SER-ASN-VAL-ASP-GLN-SER-ALA-LEU which corresponds to the residues 119 to 129 of the polypeptide chain. To define which of the two serines is phosphorylated, the present experiments were carried out. The 32P-labeled chymotryptic fragment was obtained from alpha crystallin isolated from the outer cortex of calf lenses incubated in the presence of [32P]-orthophosphate. By analyses of the products obtained after Edman degradation, utilizing electrophoresis in cellulose TLC plates and radioautography, it was possible to locate the phosphate in the serine residue at position 122 in the polypeptide chain. No phosphate could be detected in the serine residue at position 127.

The photochemistry of specific tryptophan residues in proteins as analyzed by the fluorescent scanning of tryptic peptide maps.

Abstract— The fact that most proteins contain several tryptophans hinders the investigation of the photochemistry of a particular indole residue. A method is presented here that can be used to investigate the photochemistry of specific tryptophan residues in proteins. It consists simply of separating the peptides of a proteolytically digested protein by TLC and then scanning the peptides at the fluorescent maximum of tryptophan. The assignment of the resultant peaks to a particular peptide is based on the chromatographic comparison of the scans with peptide maps.