Synuclein proteins of the pufferfish Fugu rubripes: sequences and functional characterization

In humans, three genes encode the related alpha-, beta-, and gamma-synucleins, which function as lipid-binding proteins in vitro. They are being widely studied, mainly because of the central involvement of alpha-synuclein in a number of neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In these diseases, the normally soluble alpha-synuclein assembles into abnormal filaments. Here, we have identified and characterized the synuclein gene family from the pufferfish Fugu rubripes. It consists of four genes, which encode alpha-, beta-, gamma1-, and gamma2-synucleins. They range from 113 to 127 amino acids in length and share many of the characteristics of human synucleins, including the presence of imperfect amino-terminal repeats of 11 amino acids, a hydrophobic middle region, and a negatively charged carboxy-terminus. All four synucleins are expressed in the Fugu brain. Recombinant Fugu synucleins exhibited differential liposome binding, which was strongest for alpha-synuclein, followed by beta-, gamma2-, and gamma1-synucleins. In assembly experiments, Fugu alpha-, gamma1-, and gamma2-synucleins formed filaments more readily than human alpha-synuclein. Fugu beta-synuclein, by contrast, failed to assemble in bulk. Filament assembly of synucleins was directly proportional to their degree of hydrophobicity and their tendency to form beta-sheet structure, and correlated inversely with their net charge.     

Synucleins--to have or not to have

Synucleins, a protein family little known even three years ago, became extremely popular after two discoveries. First, alpha-synuclein was found to be involved in etiology and pathogenesis of neurodegenerative disorders. Second, some newly discovered synucleins were found to participate in development and function of certain divisions of the nervous system and some other tissues, as well as in malignisation of breast tumors. It is now evident that synucleins are a fundamentally new group of proteins. Despite the striking similarity of their amino-acid sequences, they have diverse and multiple functions. An important challenge for biomedical science is to understand functions of sinucleins in normal cells and their role in pathology.     

cDNA sequences of two apolipoproteins from lamprey

The messages for two small but abundant apolipoproteins found in lamprey blood plasma were cloned with the aid of oligonucleotide probes based on amino-terminal sequences. In both cases, numerous clones were identified in a lamprey liver cDNA library, consistent with the great abundance of these proteins in lamprey blood. One of the cDNAs (LAL1) has a coding region of 105 amino acids that corresponds to a 21-residue signal peptide, a putative 8-residue propeptide, and the 76-residue mature protein found in blood. The other cDNA (LAL2) codes for a total of 191 residues, the first 23 of which constitute a signal peptide. The two proteins, which occur in the "high-density lipoprotein fraction" of ultracentrifuged plasma, have amino acid compositions similar to those of apolipoproteins found in mammalian blood; computer analysis indicates that the sequences are largely helix-permissive. When the sequences were searched against an amino acid sequence data base, rat apolipoprotein IV was the best matching candidate in both cases. Although a reasonable alignment can be made with that sequence and LAL1, definitive assignment of the two lamprey proteins to typical mammalian classes cannot be made at this point.     

Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins.

The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous alpha-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, beta- and gamma-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. beta-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas alpha- and gamma-synucleins were slightly more compact and structured. gamma-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to alpha-synuclein, beta- And gamma-synucleins inhibit alpha-synuclein fibril formation. Complete inhibition of alpha-synuclein fibrillation was observed at 4:1 molar excess of beta- and gamma-synucleins. No significant incorporation of beta-synuclein into the fibrils was detected. The lack of fibrils formed by beta-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed.     

Chaperone-like activity of synucleins

Synucleins are a family of small proteins that are predominantly expressed in neurons. The functions of the synucleins are not entirely understood, but they have been implicated in the pathogenesis of several neurodegenerative diseases. Our data show that alpha-, beta- or gamma-synuclein suppresses the aggregation of thermally denatured alcohol dehydrogenase and chemically denatured insulin. The A53T but not the A30P mutant alpha-synuclein was able to inhibit the aggregation of insulin and the chaperone-like activity of alpha-synuclein was lost upon removal of its C-terminal residues 98-140. These results demonstrate that synucleins with the exception of the A30P mutant possess chaperone-like activity.     

The role of alpha-synuclein in the development of the dopaminergic neurons in the substantia nigra and ventral tegmental area

Alpha-synuclein is a presynaptic protein of vertebrates that belongs to the family of synucleins. Normal functions of synucleins remain unknown. Alpha-synuclein is one of the causative factors of the familial and idiopathic forms of Parkinson’s disease (PD). The progressive loss of dopaminergic (DA) neurons is characteristic of PD and the most severe damage occurs in the substantia nigra (SN). This leads to an erraticism of the synthesis and synaptic secretion of the neurotransmitters, subsequently resulting in the loss of the connections between brain areas. This work shows that alpha-synuclein is directly involved in the formation of the mature DA neurons of the midbrain at different stages of the ontogenesis and these findings are consistent with data obtained in other studies. Thus, alpha-synuclein may have a varying modulating effect on the growth dynamics and the fate of populations of DA neurons.     

Complete sequence of the lamprey fibrinogen alpha chain

The complete amino acid sequence of the lamprey fibrinogen alpha chain has been determined by a combination of peptide sequencing and cDNA and genomic cloning. The chain, which has an apparent molecular weight by dodecyl sulfate-polyacrylamide gel electrophoresis of ca. 100,000, is composed of 961 amino acid residues and has a calculated molecular weight of 96,722. It is distinguished by a large number of 18-residue repeats in a region where mammalian fibrinogens have 13-residue repeats. The data are in accord with our previous finding that the lamprey alpha chain has a distinctive amino acid composition, almost half the residues being glycine, serine, or threonine. The chain differs from mammalian alpha chains in that there are no cysteines in the carboxy-terminal half, and thus no intrachain loop, nor are there any RGD sequences in the lamprey alpha chain. Taken together with previous data on the sequences of the beta and gamma chains, the findings bear significantly on our understanding of fibrin formation. The alpha chain also provides an interesting case of structural convergence during evolution.     

alpha-synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356.

alpha-Synuclein has been implicated in the pathogenesis of several neurodegenerative disorders based on the direct linking of missense mutations in alpha-synuclein to autosomal dominant Parkinson's disease and its presence in Lewy-like lesions. To gain insight into alpha-synuclein functions, we have investigated whether it binds neuronal proteins and modulates their functional state. The microtubule-associated protein tau was identified as a ligand by alpha-synuclein affinity chromatography of human brain cytosol. Direct binding assays using (125)I-labeled human tau40 demonstrated a reversible binding with a IC(50) about 50 pM. The interacting domains were localized to the C terminus of alpha-synuclein and the microtubule binding region of tau as determined by protein fragmentation and the use of recombinant peptides. High concentrations of tubulin inhibited the binding between tau and alpha-synuclein. Functionally, alpha-synuclein stimulated the protein kinase A-catalyzed phosphorylation of tau serine residues 262 and 356 as determined using a phospho-epitope-specific antibody. We propose that alpha-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly

Neuronal and oligodendrocytic aggregates of fibrillar alpha-synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise viability of affected cells. Here, we report that a 12-amino acid stretch ((71)VTGVTAVAQKTV(82)) in the middle of the hydrophobic domain of human alpha-synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human beta-synuclein is highly homologous to alpha-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of alpha-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of alpha-synuclein filaments, because it is resistant to proteolytic digestion in alpha-synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human alpha-synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human alpha-synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit alpha-synuclein fibrillization and might arrest disease progression.     

Human Microtubule-Associated Protein 1a (MAP1A) Gene: Genomic Organization, cDNA Sequence, and Developmental- and Tissue-Specific Expression

Microtubule-associated proteins (MAPs) regulate microtubule stability and play critical roles in neuronal development and the balance between neuronal plasticity and rigidity. MAP1a (HGMW-approved symbol MAP1A) stabilizes microtubules in postnatal axons. We describe human MAP1a's genomic organization and deduced cDNA and amino acid sequences. MAP1a is a single-copy gene spanning 10.5 kb. MAP1a coding sequence is contained in five exons. Translation begins in exon 3. Human MAP1a contains 2805 amino acids (predicted molecular weight 306.5 kDa) and is slightly larger than rat MAP1a (2774 amino acids). Like rat and bovine MAP1a, human MAP1a contains conserved tubulin binding motifs in the amino-terminal region. The carboxy-terminal portion contains a conserved pentadecapeptide that is present in the light chain portion of rat and bovine MAP1a/LC2 polyprotein. We show that human MAP1a gene expression occurs almost exclusively in the brain and that there is approximately 10-fold greater gene expression in adult brain compared to fetal brain. Strong, interspecies conservation between human and rat MAP1a cDNA and amino acid sequences indicates important relationships between MAP1a's function and its primary amino acid sequence.     

Chicken synucleins: cloning and expression in the developing embryo

Synucleins comprise a family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human diseases. Mutations of alpha-synuclein has been found in several families with hereditary early-onset Parkinson's disease and accumulation of this protein in characteristic cytoplasmic inclusions is a pathohistological hallmark of several neurodegenerative diseases that have been recently classified as 'alpha;-synucleinopathies' (reviewed in Brain Res. Bull. 50 (1999) 465; J. Neurosci. Res. 58 (1999) 120; Philos. Trans. R. Soc. Lond. Biol. Sci. 354 (1999) 1101; Brain Pathol. 9 (1999) 733). Aggregates of beta-synuclein and persyn (gamma-synuclein) also have been found in dystrophic neurites associated with Parkinson's and other neurodegenerative diseases (Proc. Natl. Acad. Sci. USA 96 (1999) 13450; and our unpublished observations). Moreover, persyn has been implicated in malignization of breast tumours (Cancer Res. 57 (1997) 759; Cancer Res. 59 (1999) 742; Hum. Mol. Genet. 7 (1998) 1417). All synucleins have distinct, although overlapping, patterns of expression in the embryonic, postnatal and adult mammalian nervous systems, suggesting important, although still not clear, biological functions in neuronal developing. Chicken embryo is a unique object for developmental studies that allows in vivo manipulations not always possible for mammalian embryos. Studies of synucleins expression in this model system could shed light on their functions in the developing nervous system. We cloned three chicken synucleins from the embryonic neural cDNA libraries and studied their expression in normal chicken embryonic tissues by Northern and in situ hybridization with specific probes. Our results demonstrate that primary structures and expression patterns of synucleins are similar in birds and mammals, suggesting that conserved function of synucleins is important for embryonic development of vertebrates.     

Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration

Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.     

Degradative organelles containing mislocalized alpha-and beta-synuclein proliferate in presenilin-1 null neurons

Presenilin-1 null mutation (PS1 -/-) in mice is associated with morphological alterations and defects in cleavage of transmembrane proteins. Here, we demonstrate that PS1 deficiency also leads to the formation of degradative vacuoles and to the aberrant translocation of presynaptic alpha- and beta-synuclein proteins to these organelles in the perikarya of primary neurons, concomitant with significant increases in the levels of both synucleins. Stimulation of autophagy in control neurons produced a similar mislocalization of synucleins as genetic ablation of PS1. These effects were not the result of the loss of PS1 gamma-secretase activity; however, dysregulation of calcium channels in PS1 -/- cells may be involved. Finally, colocalization of alpha-synuclein and degradative organelles was observed in brains from patients with the Lewy body variant of AD. Thus, aberrant accumulation of alpha- and beta-synuclein in degradative organelles are novel features of PS1 -/- neurons, and similar events may promote the formation of alpha-synuclein inclusions associated with neurodegenerative diseases.     

Cell surface protein receptors in oral streptococci

Abstract Streptococci have a vast repertoire of adherence properties which include binding to human tissue components, epithelial cells and to other bacterial cells. These interactions are determined by the expression of cell-surface receptors some of which are species-specific. In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified. The antigen I/II family of polypeptides are wall-associated high molecular mass proteins (158–166 kDa) with several binding functions that may be attributed to different domains of the receptor molecules. The LraI family of polypeptides are surface-associated lipoproteins (32–33 kDa) involved in adherence of streptococci to salivary glycoprotein pellicle and to oral Actinomyces . A region of amino acid sequence similarity is evident amongst members of the two protein families in Streptococcus gordonii . Ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptococci within the human oral cavity.     

Evolutionary aspects of the synuclein super-family and sub-families based on large-scale phylogenetic and group-discrimination analysis

Over the last decade, many genetic studies have suggested that the synucleins, which are small, natively unfolded proteins, are closely related to Parkinson’s disease and cancer. Less is known about the molecular basis of this role. A comprehensive analysis of the evolutionary path of the synuclein protein family may reveal the relationship between evolutionarily conserved residues and protein function or structure. The phylogeny of 252 unique synuclein sequences from 73 organisms suggests that gamma-synuclein is the common ancestor of alpha- and beta-synuclein. Although all three sub-families remain highly conserved, especially at the N-terminal, nearly 15% of the residues in each sub family clearly diverged during evolution, providing crucial guidance for investigations of the different properties of the members of the superfamily. His50 is found to be an alpha-specific conserved residue (91%) and, based on mutagenesis, evolutionarily developed a secondary copper binding site in the alpha synuclein family. Surprisingly, this site is located between two well-known polymorphisms of alpha-synuclein, E46K and A53T, which are linked to early-onset Parkinson’s disease, suggesting that the mutation-induced impairment of copper binding could be a mechanism responsible for alpha-synuclein aggregation.     

The X-lectins: A new family with homology to the Xenopus laevis oocyte lectin XL-35

The Xenopus laevis oocyte cortical granule lectin (XL35) has been studied in fertilization and embryonic development. Several nucleic acid sequences that predict proteins homologous to XL35 have since been reported in frog, human, mouse, lamprey, trout, ascidian worm. These proteins also showed high degrees of amino acid sequence homology to a common fibrinogen-like motif that may involve carbohydrate binding. Although their biological functions and carbohydrate binding specificities have not been studied in detail, this new family of lectins has common characteristics. Several independent studies on this new family of lectins strongly suggest that the lectins are expressed and stored in specialized vesicles that may be released upon the infection by pathogens. In addition, some family members have been shown to bind to oligosaccharides from bacterial pathogens. Therefore, this family of lectins likely participates in pathogen surveillance as part of the innate immune system. We propose the name X-lectin family for these homologs of XL35. Published in 2004.     

The IgA-binding β antigen of the c protein complex of Group B streptococci: sequence determination of its gene and detection of two binding regions

The beta antigen of the lbc protein complex of Group B streptococci is a cell-surface receptor which binds the Fc region of human immunoglobulin A (IgA). Determination of the nucleotide sequence of the beta antigen gene shows that it encodes a preprotein having a molecular weight of 130,963 daltons and a polypeptide of 1164 amino acid residues that is typical of other Gram-positive cell-wall proteins. There is a long signal sequence of 37 amino acids at the N-terminus. Four of the five C-terminal amino acid residues are basic and are preceded by a hydrophobic stretch that appears to anchor the C-terminus in the cell membrane. To the N-terminal side of this hydrophobic stretch is a putative cell-wall-spanning region containing proline-rich repeated sequences. An unusual feature of these repeated sequences is a three-residue periodicity, whereby every first residue is a proline, the second residue is alternating positively or negatively charged, and the third residue is uncharged. The IgA-binding activity was approximately localized by expressing subfragments of the beta antigen as fusion proteins. Two distinct but adjacent DNA segments specified peptides that bound IgA, which indicates that the IgA-binding activity is located in two distinct regions of the protein.