Biomedical subjects

J Schneikert

Publications and source records attributed to J Schneikert.

5 recordsLinked to original sources

PubMed1995

Two naturally occurring mouse alpha-1,2-mannosidase IB cDNA clones differ in three point mutations. Mutation of Phe592 to Ser592 is sufficient to abolish enzyme activity.

In mammalian cells, alpha-1,2-mannosidases play an essential role in the early steps of N-linked oligosaccharide maturation. We previously reported (Herscovics, A., Schneikert, J., Athanassiadis, A., and Moremen, K. W. (1994) J. Biol. Chem. 269, 9864-9871) the isolation of mouse alpha-mannosidase IB cDNA clones from a Balb/c 3T3 cDNA library. Clone 4 encodes a type II membrane protein of 641 amino acids with a cytoplasmic tail of 35 amino acids, followed by a transmembrane domain and a large C-terminal catalytic domain, whereas clone 16 encodes only the last 471 amino acids. Their overlapping sequences (from amino acid 152) are identical, except for three point mutations that result in three amino acid differences in the catalytic domain of the enzyme (Thr411, Leu468, and Ser592 in clone 4 to Met411, Phe468, and Phe592 in clone 16, respectively). Both sequences could be amplified by polymerase chain reaction using templates of cDNAs derived from colon and brain of CD1 mice and from L cells derived from the C3H/An mouse, indicating that both are natural isoforms found in two inbred and one outbred mouse strains. When expressed in COS7 cells as a secreted protein A fusion protein, the catalytic domain of clone 16 displays alpha-1,2-mannosidase activity using [3H]mannose-labeled Man9GlcNAc as substrate, but the corresponding region of clone 4 is poorly secreted under identical conditions. The contribution of each point mutation to this differential secretion and enzyme activity of the two fusion proteins was assessed by testing the six recombinants corresponding to all the possible sequence permutations. Mutation of Phe592 to Ser592, as found in clone 4, is sufficient to abolish alpha-1,2-mannosidase activity, whereas mutation of Met411 to Thr411 or of Phe468 to Leu468 affects secretion with relatively little effect on enzyme activity. Phe592 is part of a highly conserved region that seems important for enzyme activity of class 1 alpha-1,2-mannosidases.

Amino Acid Sequence↗

PubMed1994

Characterization of a novel mouse recombinant processing alpha-mannosidase.

In previous work (Herscovics et al., J. Biol. Chem., 269, 9864-9871), a novel mouse alpha-mannosidase cDNA was isolated by homology, taking advantage of identical regions between the amino acid sequences of the yeast and rabbit liver processing alpha 1,2-mannosidases of different specificities to design degenerate oligonucleotides for reverse transcription/polymerase chain reaction. The cDNA isolated from a mouse 3T3 cDNA library encodes a 73 kDa type II membrane protein with a cytoplasmic region of approximately 35 amino acids and a large C-terminal region that contains a consensus Ca(2+)-binding sequence. To study the properties of this enzyme, the C-terminal part lacking the transmembrane region (beginning at either amino acid 106 or 171) was transiently expressed in COS cells as a secreted protein A fusion protein, and the enzymatic properties of the fusion protein bound to IgG-Sepharose were investigated. The enzyme is an alpha 1,2-mannosidase that trims Man9GlcNAc to Man5 GlcNAc (where Man is mannose and GlcNAc is N-acetyl glucosamine). The activity requires divalent cations since it is greatly inhibited by ethylene diamine tetraacetic acid (EDTA). Although Ca2+ is the most effective, the enzyme may also use Mg2+, Mn2+ or Co2+, but not Zn2+, which is inhibitory. The enzyme is inhibited by 1-deoxymannojirimycin, but not by swainsonine. We propose that this novel alpha 1,2-mannosidase cDNA encodes mouse Golgi alpha-mannosidase IB.

Animals↗

PubMed1992

Two independent activation domains in c-Ets-1 and c-Ets-2 located in non-conserved sequences of the ets gene family.

The c-Ets-1 oncoprotein is a transcription activator that specifically binds to DNA. We show, using fusion proteins with heterologous DNA-binding domains, that chicken c-Ets-1 (p68) contains two independent activation domains. The N-terminal activation domain is absent in c-Ets-1 (p54) that is generated from an alternatively spliced mRNA. A closely related member of the ets gene family, c-Ets-2, also contains two separate activation domains. They lie in the regions of the protein that are least conserved with c-Ets-1, suggesting that the activating function will determine the different physiological roles of these two proteins. The activation domains of c-Ets-1 (p68) and -2 are separated by a moderately conserved region that does not activate on its own. These sequences appear to affect stimulation by the domains, suggesting that they regulate transcription activation. Competition experiments show that c-Ets-1 and -2 interact with a common limiting coactivator. These studies provide important clues about the physiological roles of closely related members of the ets gene family.

Animals↗

PubMed1991

Repression by Jun of the Polyoma-virus enhancer overrides activation in a cell specific manner.

The activities of promoters and enhancers are generated by the combinatorial effects of the factors which interact with them. The Polyoma virus (Py) enhancer contains sequences that are positively regulated by the proto-oncogene Jun. Surprisingly, Jun has an additional and overriding repressing effect on enhancer activity, which is cell specific. Thus overall enhancer activity cannot be simply deduced from the properties of individual elements. We present evidence that repression is indirect.

Blotting, Western↗

PubMed1990

Oncogene v-jun modulates DNA replication.

Cell transformation leads to alterations in both transcription and DNA replication. Activation of transcription by the expression of a number of transforming oncogenes is mediated by the transcription factor AP1 (Herrlich & Ponta, 1989; Imler & Wasylyk, 1989). AP1 is a composite transcription factor, consisting of members of the jun and fos gene-families. c-jun and c-fos are progenitors of oncogenes, suggestion that an important transcriptional event in cell transformation is altered activity of AP1, which may arise either indirectly by oncogene expression or directly by structural modification of AP1. We report here that the v-jun oncogene and its progenitor c-jun, as fusion proteins with the lex-A-repressor DNA binding domain, can activate DNA replication from the Polyoma virus (Py) origin of replication, linked to the lex-A operator. The transcription-activation region of v-jun is required for activation of replication. When excess v-jun is expressed in the cell, replication is inhibited or 'squelched'. These results suggest that one consequence of deregulated jun activity could be altered DNA replication and that there are similarities in the way v-jun activates replication and transcription.

Base Sequence↗