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L Overbergh

Publications and source records attributed to L Overbergh.

46 records · Page 3Linked to original sources
PubMed1994

Identification of four genes coding for isoforms of murinoglobulin, the monomeric mouse alpha 2-macroglobulin: characterization of the exons coding for the bait region.

Murinoglobulins are the single chain members of the alpha 2-macroglobulin family of proteinase inhibitors in the mouse. DNA clones representing the genes coding for four different murinoglobulins were isolated from three independent mouse genomic DNA libraries. Sequence analysis demonstrated that in each gene two exons are coding for the bait region. This is the specific protein sequence in each alpha-macroglobulin, which is functionally important since it is extremely sensitive to cleavage by different proteinases. The molecular data established the existence of at least four different murinoglobulin genes. Three of these corresponded to the three cDNA clones previously identified. Sequencing of intron-exon boundaries and intron sizing allowed us to construct physical maps of the region from exon 15 to exon 25 (numbered in comparison to mouse alpha 2-macroglobulin) in each murinoglobulin gene. Southern blotting of genomic DNA from five different mouse strains confirmed this analysis and even suggested the possible existence of a fifth murinoglobulin gene. These data indicate that the mouse presents a genetic repertoire of the alpha 2-macroglobulin family much more complex than originally anticipated. The bait region exon sequences showed a considerably higher degree of divergence (72 to 88% sequence identity) than that of the flanking exon sequences coding for adjacent, structural domains of the murinoglobulin proteinase inhibitors (91 to 96%). Even more surprising was that adjacent intron sequences are conserved as faithfully as the nonbait region coding exons (90 to 96%). These data demonstrate a unique property of the bait region coding sequences, as they apparently are allowed to mutate considerably. This divergency must then confer divergent proteinase inhibitory properties to the resulting proteins.

3T3 Cells↗
PubMed1994

Distribution of mRNA coding for alpha-2-macroglobulin, the murinoglobulins, the alpha-2-macroglobulin receptor and the alpha-2-macroglobulin receptor associated protein during mouse embryogenesis and in adult tissues.

The distribution of mRNA coding for the members of the wide-spectrum proteinase scavenging system of the alpha-2-macroglobulin family was examined in the mouse: Alpha-2-macroglobulin (MAM), the murinoglobulins (MUG), the alpha-2-macroglobulin receptor (alpha 2MR) and the receptor associated protein, the heparin binding protein-44 (alpha 2MRAP/HBP-44), a component of unknown function. The results demonstrate that MAM is expressed in the mouse embryo exclusively in the liver and not before day 13 of gestation. MUG mRNA was never detected during embryogenesis. On the other hand, both the alpha 2MR and the alpha 2MRAP/HBP-44 messages were present throughout all embryonal stages examined. The distribution of the alpha 2MR mRNA was widespread in most tissues, with stronger signals observed in developing mouse brain, in whisker follicles and in the perifollicular mesenchyme, in lung, liver, kidney, intestine and placenta. The alpha 2MRAP/HBP-44 mRNA was detected predominantly in brain, lung, liver, kidney and placenta. Interestingly, within each tissue the cellular distribution of the alpha 2MR and alpha 2MRAP/HBP-44 mRNA was quite different with the most remarkable extremes observed in kidney and in placenta. The implication of these observations for receptor expression and function are discussed. Northern analysis of adult tissues extended these observations: major signals for MAM and MUG were seen only in liver, while the expression of the alpha 2MR and the alpha 2MRAP/HBP-44 was widespread with highest levels of the 15-kb alpha 2MR mRNA in liver. Kidney was the most abundant source of alpha 2MRAP/HBP-44 mRNA with the 1.8- and 3.6-kb mRNAs, derived from the same gene by alternative mRNA splicing, present in nearly constant ratios in most tissues, except in testis. The notable absence of expression of MAM in the first half of gestation indicates that during this period the receptor is scavenging for proteinases complexed to MAM derived from the maternal circulation or is being used for endocytosis of the other documented ligands, such as plasminogen activator complexes or apolipoprotein E-containing lipoprotein particles.

Animals↗
PubMed1993

Molecular cloning and sequencing of the murine alpha-2-macroglobulin receptor cDNA.

We have molecularly cloned and sequenced the mouse alpha-2-macroglobulin receptor cDNA. The cDNA contained 14849 bases with one large open reading frame of 4545 codons which is one more than in the corresponding human cDNA. Comparison of the predicted mouse and human receptor proteins revealed the very conserved nature of this receptor with an overall amino acid identity of more than 97%. A dramatic example of this is the presence of 331 cysteine residues predicted in the mouse protein, of which 327 are positionally conserved relative to human.

Amino Acid Sequence↗
PubMed1992

The primary sequence and the subunit structure of mouse alpha-2-macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.

Mouse plasma alpha-2-macroglobulin (m alpha 2M) was isolated and the N-terminal amino-acid sequences determined after separation of the 165-kDa and 35-kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5 kb expressed predominantly in liver. The smaller subunit, with the N-terminal sequence DLSSSDLT, comprises the C-terminal 257 residues of m alpha 2M and is derived from a single-chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N-glycosylation sites. The synthesis of m alpha 2M in adult liver was demonstrated by Northern blotting and in fetal liver by in-situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of m alpha 2M in the culture medium. In plasma the level of m alpha 2M was found to be stable as expected for the murine counterpart of human plasma alpha-2-macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of the murinoglobulins.

Proteinase inhibitors of the alpha 2-macroglobulin (alpha 2M) type, although well characterized in vitro, still evade a precise description of their actual role in vivo. The main reason for this is the absence of any clinical evidence for the malfunctioning of alpha 2M in humans. Moreover, despite their ubiquitous presence in animals of very different taxa, animal models are notoriously absent in this field. With the advent of transgenic animals an important tool became available in this respect. As a first step in this direction we are analyzing at the molecular level all the members of this proteinase inhibitor family in the mouse. To retrieve related sequences we screened a mouse liver cDNA library with human alpha 2M cDNA. The sequences from two isolated clones partially coded for a protein with a high degree of sequence identity with human alpha 2M, rat alpha 2M, and rat alpha 1I3. Protein sequence data from the large and small subunits of mouse alpha 2M and of the protein isolated from mouse plasma allowed us to designate the clones as coding for murinoglobulin (MUG), an alpha 2M-related single-chain proteinase inhibitor. Rescreening resulted in the isolation of 24 clones, of which 21 were related or identical to the original MUG clones. Restriction analysis led to three groups of clones of which representative members were sequenced. Two highly homologous cDNA sequences were derived, coding for proteins that displayed the typical features of alpha 2M-type proteinase inhibitors: the overall size, the positions of a putative bait region and of the internal thiol ester, and the positional conservation of cysteine residues and putative asparagine-glycosylation sites. A third related member for which only one incomplete cDNA clone was obtained and sequenced, proved to be aberrant: the bait region contained what appeared to be an intron which escaped proper splicing. A second apparent intron was present at the 5' end of the cDNA while a frameshift mutation near the 3' end (insertion of a G) caused premature termination of the reading frame when compared to the other MUG sequences. These features were confirmed from an isolated genomic clone and extended at the genomic level: the corresponding gene, a transcriptionally weakly active pseudogene, contained the small intron but as part of a larger intron. The presence of suitable intron/exon splice sites show that a relatively small part of the intron is being introduced as an exon in the mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗