Abstract
Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis.
Highlights
The N-acetylglucosaminyltransferase MGAT1 (GlcNAc-TI or GnT-1) catalyzes the transfer of GlcNAc from UDP-GlcNAc to Man5GlcNAc2Asn of glycoproteins in the medial Golgi to initiate the synthesis of complex and hybrid N-glycans (Robertson et al, 1978; Tabas et al, 1978; Kornfeld and Kornfeld, 1985)
Constructs were transfected into Chinese hamster ovary (CHO) cells and stable populations selected for hygromycin resistance were examined for resistance to the toxicity of Phaseolus vulgaris leukoagglutinin (L-PHA), and/or binding of the lectin Galanthus nivalis agglutinin (GNA)
In this paper we show that the MGAT1 inhibitory activity of GnT1IP-L requires its luminal domain
Summary
The N-acetylglucosaminyltransferase MGAT1 (GlcNAc-TI or GnT-1) catalyzes the transfer of GlcNAc from UDP-GlcNAc to Man5GlcNAc2Asn of glycoproteins in the medial Golgi to initiate the synthesis of complex and hybrid N-glycans (Robertson et al, 1978; Tabas et al, 1978; Kornfeld and Kornfeld, 1985). The longer transcript encodes a membranebound protein that inhibits MGAT1 in transfected cells, and is termed GlcNAcT-I Inhibitory Protein, Long form, GnT1IP-L (Huang and Stanley, 2010). A rat testis membrane-bound form has been termed GL54D but its activity has not been determined (Au et al, 2015). The mouse homologue of GL54D is the shorter transcript, previously termed GnT1IP-S (Huang and Stanley, 2010), and recently designated MGAT4D by the Human Genome Nomenclature Committee. When the N-terminus of GnT1IP-S is extended by a Myc or HA tag, it becomes membrane-bound and inhibits MGAT1 in cultured cells, similar to GnT1IP-L. In male germ cells mouse GnT1IP-S is probably membrane-bound like its rat homologue GL54D (Au et al, 2015). The sequence of GnT1IP-L (Genbank accession HM067443) is identical to GnT1IP-S with an additional 44 N-terminal amino acids
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