Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• The cation-dependent mannose 6-phosphate receptor plays a crucial role in lysosomal enzyme targeting.
• Understanding the receptor's structure and gene location is vital for comprehending intracellular trafficking pathways.

Purpose of the Study:

• To isolate and characterize complementary DNA (cDNA) clones encoding the human cation-dependent mannose 6-phosphate receptor.
• To determine the nucleotide sequence and deduce the amino acid sequence of the receptor.
• To identify the chromosomal location of the gene responsible for this receptor.

Main Methods:

• Isolation of cDNA clones from a human placenta lambda gt11 library.
• Nucleotide sequencing of the cDNA insert to determine the open reading frame and untranslated regions.
• Amino acid sequencing of N-terminus and tryptic peptides for sequence verification.
• Hydrophobicity profile analysis to predict membrane topology.
• Somatic cell hybrid analysis to map the gene to a specific human chromosome.

Main Results:

• Isolation of a 2463-base-pair cDNA clone for the human cation-dependent mannose 6-phosphate receptor.
• Deduction of a 277-amino acid sequence, including a 20-amino acid signal peptide and a single membrane-spanning domain.
• Identification of five potential N-glycosylation sites in the N-terminal extracytosolic domain.
• Localization of the receptor gene to human chromosome 12.

Conclusions:

• The human cation-dependent mannose 6-phosphate receptor is synthesized as a precursor with a signal peptide.
• The receptor possesses a transmembrane domain, with distinct N-terminal and C-terminal regions exposed to different cellular compartments.
• The gene encoding the cation-dependent mannose 6-phosphate receptor is located on human chromosome 12, providing a key piece of information for genetic studies.