Research Article: The short mRNA isoform of the immunoglobulin superfamily, member 1 gene encodes an intracellular glycoprotein

Date Published: July 7, 2017

Publisher: Public Library of Science

Author(s): Ying Wang, Emilie Brûlé, Tanya Silander, Beata Bak, Sjoerd D. Joustra, Daniel J. Bernard, Arun Rishi.


Mutations in the immunoglobulin superfamily, member 1 gene (IGSF1/Igsf1) cause an X-linked form of central hypothyroidism. The canonical form of IGSF1 is a transmembrane glycoprotein with 12 immunoglobulin (Ig) loops. The protein is co-translationally cleaved into two sub-domains. The carboxyl-terminal domain (CTD), which contains the last 7 Ig loops, is trafficked to the plasma membrane. Most pathogenic mutations in IGSF1 map to the portion of the gene encoding the CTD. IGSF1/Igsf1 encodes a variety of transcripts. A little studied, but abundant splice variant encodes a truncated form of the protein, predicted to contain the first 2 Ig loops of the full-length IGSF1. The protein (hereafter referred to as IGSF1 isoform 2 or IGSF1-2) is likely retained in most individuals with IGSF1 mutations. Here, we characterized basic biochemical properties of the protein as a foray into understanding its potential function. IGSF1-2, like the IGSF1-CTD, is a glycoprotein. In both mouse and rat, the protein is N-glycosylated at a single asparagine residue in the first Ig loop. Contrary to earlier predictions, neither the murine nor rat IGSF1-2 is secreted from heterologous or homologous cells. In addition, neither protein associates with the plasma membrane. Rather, IGSF1-2 appears to be retained in the endoplasmic reticulum. Whether the protein plays intracellular functions or is trafficked through the secretory pathway under certain physiologic or pathophysiologic conditions has yet to be determined.

Partial Text

Loss-of-function mutations in the immunoglobulin superfamily, member 1 gene (IGSF1/Igsf1) cause central hypothyroidism in humans (OMIM #300888) and mice [1–3]. IGSF1 is abundantly expressed in the developing and adult pituitary gland [3–5]. According to observations in both humans and mice, IGSF1-deficiency is associated with impaired hypothalamic thyrotropin-releasing hormone (TRH) stimulation of thyroid-stimulating hormone (TSH) synthesis and/or secretion by thyrotrope cells of the anterior pituitary [2, 3, 6, 7]. IGSF1’s normal function in cells and how its absence leads to impaired TRH action are presently unknown.

To enable investigations of the IGSF1-2 protein isoform, we cloned the murine cDNA into an expression vector that added a Myc/His tag to the C-terminus. This was necessary as antibodies against the N-terminus of IGSF1 produced inconsistent results (data not shown). When expressed in CHO cells, murine IGSF1-2 migrated as a single protein species of ~28 kDa (Fig 1A, lane 2). The previously described IGSF1-NTD and—CTD are glycoproteins [11]. IGSF1-2 is similarly predicted to be glycosylated at an asparagine residue (Asn43) in the first of its 2 Ig loops. Consistent with this idea, removal of N-linked sugars with either PNGaseF or EndoH hastened migration of the protein on SDS-PAGE (Fig 1A; lanes 3 and 4). Moreover, mutation of Asn43 to Gln (N43Q) caused a similar increase in IGSF1-2’s mobility (lane 5), which was not further altered by PNGaseF or EndoH (lanes 6 and 7). Collectively, these data indicate that murine IGSF1-2 is a glycoprotein, which is glycosylated at Asn43. Moreover, the equivalent effects of EndoH and PNGaseF suggest that the protein only acquires immature sugars and may therefore not transit from the ER to the Golgi in the secretory pathway.




0 0 vote
Article Rating
Notify of
Inline Feedbacks
View all comments