Expression Cloning of a Novel Suppressor of the Lec15 and Lec35 Glycosylation Mutations of Chinese Hamster Ovary Cells

doi:10.1074/jbc.271.24.13935

Expression Cloning of a Novel Suppressor of the Lec15 and Lec35 Glycosylation Mutations of Chinese Hamster Ovary Cells*

Felecia E. Ware and
Mark A. Lehrman^‡

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

^‡ To whom correspondence should be addressed:
Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041.
Tel.: 214-648-2323; Fax: 214-648-2971; E-mail: Lehrman{at}UTSW.SWMED.EDU

Abstract

Lec15 and Lec35 are recessive Chinese hamster ovary (CHO) cell glycosylation mutations characterized by inefficient synthesis and utilization, respectively, of mannose-P-dolichol (MPD). Consequently, Lec15 and Lec35 cells accumulate Man₅GlcNAc₂-P-P-dolichol and glucosaminyl-acylphosphatidylinositol. This report describes the cloning of a suppressor (termed SL15) of the Lec15 and Lec35 mutations from a CHO cDNA library by functional expression in Lec15 cells, employing phytohemagglutinin/swainsonine selection.

The SL15 protein has a predicted molecular weight of 26,693 with two potential membrane spanning regions and a likely C-terminal endoplasmic reticulum retention signal (Lys-Lys-Glu-Gln). Lec15 cells transfected with SL15 have normal levels of MPD synthase activity in vitro and convert Man₅GlcNAc₂-P-P-dolichol to Glc_0-3Man₉GlcNAc₂-P-P-dolichol in vivo. Surprisingly, SL15 also corrects the defective mannosylation in Lec35 cells. The SL15 protein bears no apparent similarity to Saccharomyces cerevisiae MPD synthase (the DPM1 protein), but is highly similar to the hypothetical F38E1.9 protein encoded on Caenorhabditis elegans chromosome 5. These results indicate a novel function for the SL15 protein and suggest that MPD synthesis is more complex than previously suspected.

Footnotes

↵* This work was generously supported by National Institutes of Health Grant GM38545 and Robert Welch Foundation Grant I-1168. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U55387[GenBank].
↵¹ The abbreviations used are:

MPD

mannose-phosphate-dolichol

MPDS

MPD synthase

CHO

Chinese hamster ovary

conA

concanavalin A

DPM1

dolichol-P-mannose synthase gene

GPD

glucose-P-dolichol

GnPPD

GlcNAc-P-P-dolichol

GPI

glycosylphosphatidylinositol

PHA

phytohemagglutinin E

Sw

swainsonine

SL15

suppressor of Lec15

kb

kilobase(s)

ER

endoplasmic reticulum

nt

nucleotide(s)

HPLC

high performance liquid chromatography.
- Received January 16, 1996.
- Revision received March 20, 1996.