Biosynthesis, Processing, and Intracellular Transport of GM2 Activator Protein in Human Epidermal Keratinocytes

Abstract

The processing, intracellular transport, and endocytosis of the G M2 activator protein (G M2 AP), an essential cofactor of ␤-hexosaminidase A for the degradation of ganglioside G M2 , was investigated in human epidermal keratinocytes. The G M2 AP precursor is synthesized as an 18-kDa peptide, which is singly glycosylated, resulting in 22-kDa high mannose and 24 -27-kDa complex glycoforms. A small portion of the 22-kDa form bears phosphomannosyl residues. About 30% of the G M2 AP precursor is secreted during 12 h after synthesis, consisting almost exclusively of complex glycoforms. In a post-Golgi compartment, the intracellular remainder is converted to a 20-kDa mature form within 24 h, bearing a heavily trimmed N-glycan on a 17-kDa backbone. Interestingly, even nonglycosylated G M2 AP is delivered to the lysosome, as shown by tunicamycin treatment and subcellular fractionation. Also, its endocytosis is independent of carbohydrate-linked signals and is even more effective for nonglycosylated G M2 AP. We conclude that a mannose-6-phosphate-independent pathway for the lysosomal delivery of G M2 AP exists in cultured human keratinocytes.

Complete localization of disulfide bonds in GM2 activator protein

“…The protein was shown to be homogeneous by MALDI-TOF MS and the molecular mass was determined to be 20,532 + 3 Da, consistent with a glycosylation of (Hex)3(HexNAc)2(Fuc) I and four disulfide bonds (theoretical mass 20,531 Da). This result also confirms the N-terminus after proteolytic removal of the signalling peptide to be His 24, as predicted previously (Glombitza et al, 1997).…”

Influence of N-Glycosylation and N-Glycan Trimming on the Activity and Intracellular Traffic of GD3 Synthase

“…N-Glycans were found essential for proper folding and cellular transport of the ␣ subunit of the lysosomal ␤-hexosaminidase A (31), while lysosomal delivery of GM2 activator protein, a singly glycosylated protein involved in glycolipid catabolism, was not impaired by tunicamycin (32). On the other hand, inhibition of glycosylation did not affect activity, but affected intracellular transport of secretory proteins as the platelet-derived growth factor B-chain (33), or plasma membrane proteins as the norepinephrine (34) and GLYT1 glycine (35) transporters.…”

The Possible Contribution of a General Glycosphingolipid Transporter, GM2 Activator Protein, to Atherosclerosis

“…A deficiency of GM2 activator protein results in the storage of GM2 ganglioside and severe neurological disease, Sandhoff disease 5) . The GM2 activator protein is synthesized as a 22-kDa precursor bearing a single high mannose N-linked oligosaccharide chain on a peptide backbone of 18 kDa in the rough endoplasmic reticulum 30) . In the Golgi apparatus, at least 70% of the 22-kDa precursor is converted to 24-kDa precursor by remodeling N-glycan to a complex-type oligosaccharide 30) .…”

“…The GM2 activator protein is synthesized as a 22-kDa precursor bearing a single high mannose N-linked oligosaccharide chain on a peptide backbone of 18 kDa in the rough endoplasmic reticulum 30) . In the Golgi apparatus, at least 70% of the 22-kDa precursor is converted to 24-kDa precursor by remodeling N-glycan to a complex-type oligosaccharide 30) . About one-third of the precursor is secreted, and more than 90% of these secretary forms consist of the 24-kDa precursor 30) .…”