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Structure and Function of Potential Glycosylation Sites of Dynactin-Associated Protein dynAP

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Abstract

Overexpression of human dynactin-associated protein (dynAP) transforms NIH3T3 cells. DynAP is a single-pass transmembrane protein with a carboxy-terminal region (amino acids 135–210) exposed to the outside of the cell possessing one potential N-glycosylation site (position 143) and a distal C-terminal region (residues 173–210) harboring a Thr/Ser-rich (T/S) cluster that may be O-glycosylated. In SDS–PAGE, dynAP migrates anomalously at ~ 45 kDa, much larger than expected (22.5 kDa) based on the amino acid composition. Using dynAP mutants, we herein showed that the T/S cluster region is responsible for the anomalous migration. The T/S cluster region is required for transport to the cytoplasmic membrane and cell transformation. We produced and purified the extracellular fragment (dynAP135–210) in secreted form and analyzed the attached glycans. Asn143 displayed complex-type glycosylation, suggesting that oligosaccharide transferase may recognize the NXT/S sequon in the secretory form, but not clearly in full-length dynAP. Core I-type O-glycosylation (Gal-GalNAc) was observed, but the mass spectrometry signal was weak, clearly indicating that further studies are needed to elucidate modifications in this region.

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Data Availability

All relevant data can be found in the paper and its Supporting Information files. MS data are available in the GlycoPOST repository (http://glycopost.glycosmos.org/) as GlyToucan ID GPST000172, GPST000173, and GPST000174.

Abbreviations

3-AQ:

3-Aminoquinoline

DAPI:

4′,6-Diamidino-2-phenylindole

DMEM:

Dulbecco’s Modified Eagle Medium

dynAP:

Dynactin-associated protein

GFP:

Green fluorescent protein

MS:

Mass spectrometry

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TM:

Transmembrane

TFA:

Trifluoroacetic acid

TBS:

Tris-buffered saline

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Funding

This work was supported by JSPS KAKENHI Grant Number 24300343 and Daiichi Sankyo Company.

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Authors and Affiliations

  1. Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga, 526-0829, Japan

    Xiaobo Yin, Takayuki Konishi, Kazuo Horikawa, Ryota Tanaka, Yuki Togo, Takanori Noda, Miho Hosoi, Mie Tsuchida, Tatsuki Kunoh, Shuichi Wada, Toshinobu Nakamura, Ryuzo Sasaki, Tamio Mizukami & Makoto Hasegawa

  2. Frontier Pharma, 1281-8 Tamura-cho, Nagahama, Shiga, 526-0829, Japan

    Miho Hosoi, Mie Tsuchida, Ryuzo Sasaki & Tamio Mizukami

  3. Specialty Medicine Research Laboratories 1, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan

    Eisuke Tsuda

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  1. Xiaobo Yin
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Correspondence to Makoto Hasegawa.

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Yin, X., Konishi, T., Horikawa, K. et al. Structure and Function of Potential Glycosylation Sites of Dynactin-Associated Protein dynAP. Mol Biotechnol 64, 611–620 (2022). https://doi.org/10.1007/s12033-021-00435-3

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