Abstract
A novel human apolipoprotein designated apolipoprotein M (apoM) is described. The unique N-terminal amino acid sequence of apoM was found in an approximately 26-kDa protein present in a protein extract of triglyceride-rich lipoproteins (TGRLP). The isolated apoM cDNA (734 base pairs) encoded a 188-amino acid residue-long protein, distantly related to the lipocalin family. The mRNA of apoM was detected in the liver and kidney. Western blotting demonstrated apoM to be present in high density lipoprotein (HDL) and to a lesser extent in TGRLP and low density lipoproteins (LDL). The first 20 amino acid residues of apoM constituted a hydrophobic segment with characteristic features of a signal peptide. This was retained in the mature protein because of the lack of a signal peptidase cleavage site. In vitro translation in the presence of microsomes demonstrated translocation of apoM over the membrane and glycosylation but no signal peptide cleavage. The in vitro translated product remained associated with the microsomes after treatment with carbonate at pH 11, demonstrating that apoM is an integral protein. This finding suggests that apoM is linked to the single phospholipid layer of lipoproteins with a hydrophobic signal anchor. In conclusion, a novel human apolipoprotein, the function of which remains to be determined, is described.
Highlights
The nucleotide sequence and deduced protein sequence in this paper can be accessed through NCBI Protein Database under NCBI accession number AF118393
ApoB-100 is the structural apolipoprotein of low density lipoproteins (LDL), IDL, and very low density lipoproteins (VLDL), whereas apoB-48 plays this role in chylomicron [1,2,3]
Identification and Cloning of a Novel Apolipoprotein—A previously unrecognized amino acid sequence (MFHQIWAALLYFYGI) was identified when a 26-kDa protein in TG-rich lipoproteins (TGRLP) was analyzed by N-terminal sequence
Summary
Materials—A human liver 5Ј-stretch plus cDNA library, a human liver Marathon-ready cDNA library, human multiple-tissue Northern blot membranes, Zoo blot membranes, and Express Hyb solution were purchased from CLONTECH. No homologous protein was identified, but several human expressed sequence tags [13] were found to predict the amino acid sequence. Based on their sequences, two oligonucleotides (oligo-1 and oligo-2) corresponding to the first or second strand of the sequence 5Ј-ATG TTC CAC CAA ATT TGG GCG GCT CTG CTC TAC TTC TAT GGT ATT were synthesized. To determine the relative amount of apoM in lipoprotein-deficient plasma, increasing amounts of normal plasma (0.75, 1.25, 2.5, 5, 10, and 20 g of plasma proteins) were applied on SDS-polyacrylamide gels and analyzed by Western blotting using the anti-peptide antiserum against apoM. A prestained radiolabeled standard mixture was used as molecular weight marker
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