Isolation and characterization of the MHC linked β‐type proteasome subunit MC13 cDNA

Frentzel, Stefan; Gräf, Uwe; Hämmerling, Günter J.; Kloetzel, Peter M.

doi:10.1016/0014-5793(92)80420-l

Cited by 28 publications

(11 citation statements)

References 22 publications

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“…The complete nucleotide sequence of the mouse Lmp-7 gene is displayed in Figure 2. The exon/intron boundaries are consistent with the published sequence of the mouse Lmp-7 cDNA (MC13; Frentzel et al 1992) and a cDNA clone previously isolated in our laboratory (Zanelli and co-workers, unpublished data). 1; Trowsdale et al 1991).…”

Section: Isolation and Restriction Map Of The Mouse Lmp-7 Genesupporting

confidence: 87%

Genomic organization and tissue expression of the mouse proteasome gene Lmp-7

Zanelli¹,

Zhou²,

Cao³

et al. 1993

Immunogenetics

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LMP7 is one of the two proteasome subunits encoded in the major histocompatibility complex and is speculated to play a role in the generation of endogenous peptides for presentation by class I molecules to cytotoxic T cells. Here we report the genomic organization of the mouse Lmp-7 gene and the tissue distribution of its messenger RNA. In contrast to human LMP7 which is composed of seven exons and six introns, the mouse Lmp-7 gene is organized in six exons and five introns. Interestingly, the region corresponding to the first exon of human LMP7 is highly modified by numerous insertions and deletions and contains two in frame stop codons. Consequently, the mouse Lmp-7 gene does not allow the alternative exon usage described in humans and most likely encodes for only one LMP7 protein. Thus, the Tap-1 3' end gene region and the Lmp-7 initial translation codon are separated by an 1182 nucleotide region which contains a TATA-box, a cAMP regulatory element, two SP1 sites, and two G-C-rich regions. Expression of the Lmp-7 messenger RNA was analyzed on different tissues from unstimulated mice. Lmp-7 messenger RNA is expressed in spleen, thymus, lung, liver, heart, and, at a very low level, in kidney but not in brain and testis. The possible role of Lmp genes in antigen processing is discussed.

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Section: Isolation and Restriction Map Of The Mouse Lmp-7 Genesupporting

confidence: 87%

Genomic organization and tissue expression of the mouse proteasome gene Lmp-7

Zanelli¹,

Zhou²,

Cao³

et al. 1993

Immunogenetics

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“…B8 cells were grown in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 5 ϫ 10 Ϫ5 M 2-mercaptoethanol, 100 units/ml penicillin/streptomycin, 250 g/ml G418. B8 cells were transfected with plasmids encoding BALB/c-derived LMP2 or LMP7 full-length cDNAs cloned into pSG5 (Stratagene, Heidelberg, Germany) by direct cloning using EcoRI sites for LMP7 (42) and PCR cloning using BamHI/BglII sites and primers as described for LMP2 (15). Cells (5 ϫ 10 5 ) plated to 60% confluence were transfected by standard calcium phosphate precipitation methods with 8 g of pLMP2 or 8 g of pLMP7 or 4 g of each in cotransfection with 2 g of either puromycin (pLXSP) or hygromycin (pLXSH) resistance expression constructs (generous gifts of Dr. Ed Palmer, Basel, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

The Interferon-γ-inducible 11 S Regulator (PA28) and the LMP2/LMP7 Subunits Govern the Peptide Production by the 20 S Proteasome in Vitro

Groettrup

Ruppert

Kuehn

et al. 1995

Journal of Biological Chemistry

216

179

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Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-␥, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.

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“…The rat and mouse homologues of LMW have recently been cloned [12,131. Their derived protein sequences are 90 % identical to the published LMP7 sequence (hereafter referred to as LMP7a) for most of its length.…”

Section: Introductionmentioning

confidence: 99%

The major histocompatibility complex‐encoded proteasome component LMP7: alternative first exons and post‐translational processing

et al. 1993

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The LMP7 gene maps to the major histocompatibility complex class II region. The derived protein sequence shares homology with N-terminal amino acid sequence from proteasome subunits (Glynne, R., Powis, S. H., Beck, S., Kelly, A., Kerr, L.-A. and Trowsdale, J., Nature 1991. 353: 357) and it has been suggested that LMP7 is involved in the degradation of endogenous antigens prior to their presentation through class I (Robertson, M., Nature 1991. 353: 300). We have isolated a second LMP7 transcript which has a different first exon to the published sequence. Both transcripts were expressed in cell lines from a number of tissues and both responded to interferon-gamma. An anti-LMP7 antiserum precipitated proteins similar in their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to those precipitated by an anti-proteasome serum. Western blot analysis of anti-proteasome precipitates demonstrated that the LMP7 protein is incorporated into the proteasome but has a molecular mass of 23 kDa, 7 kDa smaller than expected fro the derived protein sequence of either of the cDNA. A pulse-chase experiment indicated that post-translational cleavage of the LMP7 N terminus precedes the formation of the 23-kDa proteasome subunit. To our knowledge, LMP7 provides the first biochemical evidence for such processing of proteasome components.

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Isolation and characterization of the MHC linked β‐type proteasome subunit MC13 cDNA

Cited by 28 publications

References 22 publications

Genomic organization and tissue expression of the mouse proteasome gene Lmp-7

Genomic organization and tissue expression of the mouse proteasome gene Lmp-7

The Interferon-γ-inducible 11 S Regulator (PA28) and the LMP2/LMP7 Subunits Govern the Peptide Production by the 20 S Proteasome in Vitro

The major histocompatibility complex‐encoded proteasome component LMP7: alternative first exons and post‐translational processing

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