Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein.

Abstract

Abstract. Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.T H~ majority of integral membrane glycoproteins span the membrane once and are oriented with either their carboxy-terminal (type I) or amino-terminal (type II) amino acids exposed to the cytoplasm. Type I glycoproteins typically have a cleavable signal sequence at the amino terminus, a carboxy-proximal hydrophobic membrane anchor and a carboxy-terminal hydrophilic cytoplasmic tail. Type II glycoproteins are characterized by an amino-terminal cytoplasmic tail, followed by a 20-30-amino acid hydrophobic transmembrane domain, with the remainder of the protein being oriented externally, comprising the ectodomain.The intracellular processing and transport of membrane glycoproteins has been the object of extensive study. EluciDr. Spriggs's current address is lmmunex Corp., 51 University Street, Seattle WA 98101....

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

“…In addition to their role in signal transduction, the cytoplasmic domains of surface glycoproteins frequently influence protein processing and transport (Moll et al, 2002;Parks & Lamb, 1990;Spriggs & Collins, 1990;Wilson et al, 1990). Generally, a minimal domain of 10-15 amino acids is required for type II glycoproteins to ensure correct translocation of the nascent protein chain into the ER and subsequent post-translational modifications (Parks & Lamb, 1990;Spriggs & Collins, 1990;Wilson et al, 1990).…”

“…Paramyxovirus glycoprotein functions are also regulated by intracellular processes, where the amino-terminal cytoplasmic domain serves as a signal peptide for the translocation of the nascent protein chain into the endoplasmic reticulum (ER) (Spriggs & Collins, 1990), thereby modulating protein maturation and surface transport. While the morbillivirus F and H proteins associate early during biosynthesis in the ER and are transported to the cell surface as a complex (Plemper et al, 2001), the henipavirus F and G proteins are transported independently (Whitman et al, 2009), indicating that differences in interactions between glycoproteins may influence the assembly and cell-cell fusion process.…”

Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: altered O-glycosylation in the presence of brefeldin A

“…The interval of spacing between the larger of these species was approximately 2K to 2-25K, suggesting that the incremental increases were due to single-chain increases in the number of N-linked oligosaccharides. When synthesized in parallel (not shown), the HN protein of human parainfluenza virus type 3 was glycosylated into a single species which comigrated with the authentic protein (Spriggs & Collins, 1990), suggesting that the inefficient glycosylation of the G protein was characteristic of that molecule rather than due to a deficiency of the in vitro system. The two major, largest in vitro glycosylated species (labelled C and D in Fig.…”

“…In one experiment ( Fig. 7 d and e), the immunoprocipitation step was performed first and the proteins were released by boiling in citrate buffer (see below), treated with endoglycosidase H (endo H) (Spriggs & Collins, 1990), diluted in RIPA buffer and analysed by lcetin binding.…”

“…A eDNA clone of the G mRNA cloned in the BamHI site of plasmid pGem3 (Promega) was linearized and used as a template to synthesize capped mRNA in vitro with T7 phage polymerase in the presence of GpppG. mRNA was translated in reticulocyte lysates (Promega) with [35S]methionine and, where indicated, dog pancreatic membranes (Promega) following the supplier's protocols (Spriggs & Collins, 1990).…”