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

doi:10.1074/jbc.272.8.5199

Biosynthesis, Processing, and Intracellular Transport of G_M2 Activator Protein in Human Epidermal Keratinocytes

THE LYSOSOMAL TARGETING OF THE G_M2 ACTIVATOR IS INDEPENDENT OF A MANNOSE-6-PHOSPHATE SIGNAL*

From the ^‡ Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Federal Republic of Germany and
^§ Hautklinik der Universitätskliniken Bonn, D-53127 Bonn, Federal Republic of Germany

^¶ To whom correspondence should be addressed. Tel.: 49-228-73-53-46; Fax: 49-228-73-77-78; E-mail: sandhoff{at}uni-bonn.de

Abstract

The processing, intracellular transport, and endocytosis of the G_M2 activator protein (G_M2AP), an essential cofactor of β-hexosaminidase A for the degradation of ganglioside G_M2, was investigated in human epidermal keratinocytes. The G_M2AP 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_M2AP 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_M2AP 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_M2AP. We conclude that a mannose-6-phosphate-independent pathway for the lysosomal delivery of G_M2AP exists in cultured human keratinocytes.

Footnotes

↵* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 284. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to the memory of Dr. W. Wille (1946-1992).
↵¹ The abbreviations used are:

G_M2 ganglioside

GalNAc β1 → 4Gal(3 ← 2αNeuNAc)β1 → 4Glc β1 → 1 ceramide

G_M2AP

G_M2 activator protein

BFA

brefeldin A

endo H

β-endo-N-acetylglucosaminidase H

ER

endoplasmic reticulum

hEK

human epidermal keratinocyte

β-hexosaminidase

2-acetamido-2-deoxy-β-D-hexoside acetamidodeoxyhexohydrolase (EC 3.2.1.52)

M6P

mannose-6-phosphate

PNGase F

peptide-N-glycanase F

PAGE

polyacrylamide gel electrophoresis

P_HM

precursor; high mannose

P_C

precursor; complex

P_MA

precursor with multiantennary N-glycan

M

mature G_M2AP

dP

deglycosylated G_M2AP precursor peptide

dM

deglycosylated G_M2AP mature peptide.
↵² G. J. Glombitza, and K. Sandhoff, unpublished observations.
↵³ E. Becker, G. J. Glombitza, and K. Sandhoff, unpublished procedures.
- Received September 25, 1996.
- Revision received November 19, 1996.