Margaret E. Chamberlin

Methionine Adenosyltransferase I/III Deficiency: Novel Mutationsand Clinical Variations

Methionine adenosyltransferase (MAT) I/III deficiency, caused by mutations in the MAT1A gene, is characterized by persistent hypermethioninemia without elevated homocysteine or tyrosine. Clinical manifestations are variable and poorly understood, although a number of individuals with homozygous null mutations in MAT1A have neurological problems, including brain demyelination. We analyzed MAT1A in seven hypermethioninemic individuals, to provide insight into the relationship between genotype and phenotype. We identified six novel mutations and demonstrated that mutations resulting in high plasma methionines may signal clinical difficulties. Two patients-a compound heterozygote for truncating and severely inactivating missense mutations and a homozygote for an aberrant splicing MAT1A mutation-have plasma methionine in the 1,226-1,870 microM range (normal 5-35 microM) and manifest abnormalities of the brain gray matter or signs of brain demyelination. Another compound heterozygote for truncating and inactivating missense mutations has 770-1,240 microM plasma methionine and mild cognitive impairment. Four individuals carrying either two inactivating missense mutations or the single-allelic R264H mutation have 105-467 microM plasma methionine and are clinically unaffected. Our data underscore the necessity of further studies to firmly establish the relationship between genotypes in MAT I/III deficiency and clinical phenotypes, to elucidate the molecular bases of variability in manifestations of MAT1A mutations.

Molecular characterization of Plasmodium falciparum S-adenosylmethionine synthetase

S-Adenosylmethionine (AdoMet) synthetase (SAMS: EC 2.5.1.6) catalyses the formation of AdoMet from methionine and ATP. We have cloned a gene for Plasmodium falciparum AdoMet synthetase (PfSAMS) (GenBank accession no. AF097923), consisting of 1209 base pairs with no introns. The gene encodes a polypeptide (PfSAMS) of 402 amino acids with a molecular mass of 44844 Da, and has an overall base composition of 67% A+T. PfSAMS is probably a single copy gene, and was mapped to chromosome 9. The PfSAMS protein is highly homologous to all other SAMS, including a conserved motif for the phosphate-binding P-loop, HGGGAFSGKD, and the signature hexapeptide, GAGDQG. All the active-site amino acids for the binding of ADP, P(i) and metal ions are similarly preserved, matching entirely those of human hepatic SAMS and Escherichia coli SAMS. Molecular modelling of PfSAMS guided by the X-ray crystal structure of E. coli SAMS indicates that PfSAMS binds ATP/Mg(2+) in a manner similar to that seen in the E. coli SAMS structure. However, the PfSAMS model shows that it can not form tetramers as does E. coli SAMS, and is probably a dimer instead. There was a differential sensitivity towards the inhibition by cycloleucine between the expressed PfSAMS and the human hepatic SAMS with K(i) values of 17 and 10 mM, respectively. Based on phylogenetic analysis using protein parsimony and neighbour-joining algorithms, the malarial PfSAMS is closely related to SAMS of other protozoans and plants.

Characterization of the regulatory domains of the human skn-1a/Epoc-1/Oct-11 POU transcription factor.

The Skn-1a POU transcription factor is primarily expressed in keratinocytes of murine embryonic and adult epidermis. Although some POU factors expressed in a tissue-specific manner are important for normal differentiation, the biological function of Skn-1a remains unknown. Previous in vitro studies indicate that Skn-1a has the ability to transactivate markers of keratinocyte differentiation. In this study, we have characterized Skn-1a’s transactivation domain(s) and engineered a dominant negative protein that lacked this transactivation domain. Deletional analysis of the human homologue of Skn-1a with three target promoters revealed the presence of two functional domains: a primary C-terminal transactivation domain and a combined N-terminal inhibitory domain and transactivation domain. Skn-1a lacking the C-terminal region completely lost transactivation ability, irrespective of the promoter tested, and was able to block transactivation by normal Skn-1a in competition assays. Compared with full-length, Skn-1a lacking the N-terminal region demonstrated either increased transactivation (bovine cytokeratin 6 promoter), comparable transactivation (human papillomavirus type 1a long control region), or loss of transactivation (human papillomavirus type 18 long control region). The identification of a primary C-terminal transactivation domain enabled us to generate a dominant negative Skn-1a factor, which will be useful in the quest for a better understanding of this keratinocyte-specific gene regulator.

Developmental expression of the sperm receptor of the mouse zona pellucida.

Fertilization is the culmination of a series of carefully orchestrated events that result in the formation of a one-cell zygote with the potential to develop into an adult animal.’ The molecular details of these events have been intensely investigated over the last decade, and the importance of the role of the zona pellucida at fertilization and during early development has become increasingly clear. The zona mediates the relatively species-specific binding of sperm to ovulated eggs, which results in the induction of the acrosome reaction, crucial to penetration of the zona by the capacitated sperm. Following fertilization, the zona pellucida is modified to prevent the penetration of additional sperm, and thus it acts as a major barrier to polyspermy. The zona also provides protection for the early embryo as it passes down the oviduct before implantation.’-’ Many of these molecular details have been elucidated using the mouse as an experimental system. The mouse zona pellucida can be isolated as an intact structure (FIG. 1) and is composed of three sulfated glycoproteins designated ZP1, ZP2, and ZP3, with average molecular weights of 185-200,000, 120140,000, and 83,000 daltons, respect i~ely.~.~ These proteins are synthesized during oogenesis and secreted to form an extracellular glycocalyx which surrounds the growing oocyte, ovulated egg, and dividing embryo. Specific functions have been ascribed to each protein. ZP3 contains 0-linked oligosaccharide side chains capable of inhibiting in vitro sperm-egg interactions, and the presence of at least a portion of its polypeptide backbone is necessary for the induction of the sperm acrosome reaction. ZP2 appears to act as a secondary sperm receptor, and its postfertilization modification may play a role in the block to polyspermy. ZP1, which is a dimer, may serve to cross-link copolymers of ZP2 and ZP3 and thus add structural integrity to the zona to facilitate the protection of the embryo as it passes down the o v i d u ~ t . ” ~ Although this report will concentrate on the mouse zona pellucida, it must be realized that important strides have been made investigating the zonae of a number of other species, particularly the pig and cow. We recently undertook to clone the genes that code for mouse ZP1, ZP2, and ZP3. Our results indicate that the zona genes constitute a family of single-copy genes that are located on different chromosomes and are developmentally regulated and