N-glycosylation is required for efficient secretion of a novel human secreted glycoprotein, hPAP21.
The present study reported the isolation and characterization of a novel human secreted protein, named as hPAP21 (human protease-associated domain-containing protein, 21 kDa), encoded by the hypothetical gene chromosome 2 open reading frame 7 (C2orf7) that contains signal peptide in its N-terminus, without transmembrane domain, except for PA domain in its middle. Western blotting assay indicated that the c-Myc tagged hPAP21 could be secreted into culture medium in the transfected Chinese hamster ovary cells. However, the molecular weights, whatever intracellular (28 kDa) or extracellular (30 kDa) forms, are larger than that of the prediction. To define whether the glycosylation was important process for its secretion, endoglycosidase H (Endo H) and PNGase F (PNG F) were employed to evaluate the effect of glycosylation types on secretion of hPAP21. Interestingly, the extracellular forms were primarily sensitive to PNG F, not Endo H, implying that complex N-glycosylation could be required for the secretion of hPAP21. Furthermore, N-glycosylation of Asn171 was confirmed as potential crucial process for the secretory protein via site-directed mutagenesis assay. All data will be contributed to the understanding of molecular functions of hPAP21.
Zhou
Yu-Bo
YB
College of Life Science and Biotechnology, Shanghai Jiaotong University, 1954 Huashan Road, Shanghai 200030, China.
Liu
Feng
F
Zhu
Zhi-Dong
ZD
Zhu
Hong
H
Zhang
Xin
X
Wang
Zhi-Qin
ZQ
Liu
Jian-Hua
JH
Han
Ze-Guang
ZG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002889:Chromosomes, Human, Pair 2 / Q000235:genetics*; D017124:Conserved Sequence; D006224:Cricetinae; D017931:DNA Primers; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D006023:Glycoproteins; C494126:PRADC1 protein, human