Isolation and expression of a full-length cDNA encoding the human GM2 activator protein.
We report the construction of a cDNA clone encoding a functional GM2-activator protein. The sequence of the complete 5' end of the coding region was determined by direct nucleotide sequencing of a fragment generated by multiple RACE PCR procedures from Hela cell cDNA. Specific oligonucleotides were synthesized from these data which allowed us to produce a PCR fragment that contained the complete coding sequence of the protein. This was then cloned into a mammalian expression vector. The ability of purified hexosaminidase A (beta-N-acetylhexosaminidase, EC 3.2.1.52) to hydrolyse labeled GM2 ganglioside was enhanced 10-fold more by the addition in the assay mix of lysate from transfected COS-1 cells than by the addition of identical amounts of lysate from mock transfected cells. Direct sequencing of PCR fragments from two sources also identified three polymorphisms.
Xie
B
B
Research Institute, Hospital For Sick Children, Toronto, Ontario, Canada.
McInnes
B
B
Neote
K
K
Lamhonwah
A M
AM
Mahuran
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000378:metabolism; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D049289:G(M2) Activator Protein; D015345:Oligonucleotide Probes; D021382:Protein Sorting Signals; D011506:Proteins; D011994:Recombinant Proteins; D005678:G(M2) Ganglioside; D004247:DNA