Functional Characterization of the N‐glycosylation Sites of Human Acid Sphingomyelinase by Site‐Directed Mutagenesis

Abstract

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticuIudGolgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticuludGolgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.

Sphingomyelin Phosphodiesterase Acid-like 3A (SMPDL3A) Is a Novel Nucleotide Phosphodiesterase Regulated by Cholesterol in Human Macrophages

“…SMPDL3A contains seven potential N-linked motifs, and differences in the degree of glycosylation may represent variable occupancy of these sites. N-Linked glycosylation of two discrete asparagine residues of aSMase are required for secretion and correct folding of the enzyme, and enzymatic deglycosylation of aSMase results in complete loss of activity (29). The position of both of these asparagine residues are conserved in SMPDL3A, suggesting that glycosylation may play an important role in controlling the behaviors of SMPDL3A as well.…”

“…3, indicated by bold N), with glycosylation of Asn-395 and Asn-520 being critical for normal secretion and function (29). SMPDL3A contains seven potential N-linked glycosylation motifs (defined by the pattern NX(S/T), where X is any amino acid except proline).…”

Identification and Characterization of Eight Novel SMPD1 Mutations Causing Types A and B Niemann-Pick Disease

“…A minor portion is cleaved in the endoplasmic reticulum-Golgi complex, yielding a 57-kDa form, and the majority is processed to a 70-kDa mature form (3). Six N-glycosylation sites exist in the protein, of which five are occupied (4).…”

Caenorhabditis elegansContains Two Distinct Acid Sphingomyelinases

“…There are six potential N-linked glycosylation sites (amino acids 86, 175, 335, 395, 503, and 520, respectively) in hASM. Site-directed mutagenesis studies show that five of these sites (amino acid 86, 175, 335, 395, and 520) are glycosylated in COS-1 cells (10). Elimination of any of these sites results in various degrees of decreased enzyme activity.…”