Simple, Automated, High Resolution Mass Spectrometry Method to Determine the Disulfide Bond and Glycosylation Patterns of a Complex Protein

Abstract

Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein.To enter cells and begin replication, enveloped viruses must fuse the membrane coating the viral particle with a cellular membrane to deliver a subviral particle inside the cell (for review, see Refs. 1, 2). The fusion of two membranes is thermodynamically favored but comes with a very high kinetic barrier(s). Enveloped viruses have one or more glycoproteins to mediate the fusion process using the energy liberated upon conformational changes of the viral glycoprotein(s) to clear the kinetic barrier(s). Viral entry begins when the viral glycoproteins bind an appropriate cell surface protein initiating one of three possible mechanistic fates. The viral glycoprotein-receptor interaction can serve to enable trafficking of the virion into the endocytic pathway where the low pH environment triggers a conformation change in the viral glycoproteins initiating fusion of the viral and endosome membranes (e.g. influenza virus). With other viruses, for example most retroviruses, the viral glycoprotein-receptor interaction itself triggers a conformational change in the viral glycoproteins at neutral pH at the cell surface sufficient for the ultimate fusion of the viral and cellular membranes. In a third mechanism, the interaction between the viral glycoprotein and receptor at neutral pH at the...

Reverse Engineering Provides Insights on the Evolution of Subgroups A to E Avian Sarcoma and Leukosis Virus Receptor Specificity

“…However, these residues could not be studied further since the mutant protein could not fold. In our recently published study (see below) we mapped the disulfide bond pattern of the pre-fusion form of ASLV(A) Env [36]. There are 19 Cys residues in ASLV(A) Env; one residue must be free (Figure 3).…”

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

“…However, other glycoproteins may contain one or more occupied glycosylation sites near cysteine residues involved in a disulfide bond. In such cases, a deglycosylation step must be added to the sample preparation to reduce the complexity of the MS/MS data of the disulfide bonded peptides, because glycosylation cannot be simply regarded as a modification on the peptide chains [71, 75, 77]. For example, trypsin digestion of the HIV-1 Env sequence variant, C97ZA012 gp140, which contains 25 N-linked glycosylation sites and 10 disulfide bonds, resulted in seven tryptic, disulfide-linked peptides that all contain at least one occupied glycosylation site on the disulfide-linked chains [75].…”

“…Disulfide bond mapping by profile comparison is usually done using two samples: a protein digest without reduced disulfide bonds (non-reduced sample) and a digest with reduced disulfide bonds (reduced sample). The two samples are separated in two LC runs (experiments), and peptides that are present in the ultraviolet (UV) or total ion chromatogram (TIC) profile of the non-reduced sample, but are absent in that of the reduced sample, are generally considered to be disulfide bonded, and MS/MS data can be used to assign the disulfide bonds [55, 56, 77].…”

Glycosylation of ALV-J Envelope Protein at Sites 17 and 193 Is Pivotal in the Virus Infection

“…Disulfide bonds are another important factor. Mass spectrometry indicated that in Env (ALV-A), a disulfide bond links the two subunits through the first cysteine residue Cys25 in gp85 and the last extracellular cysteine Cys438 in gp37 ( 34 ). Those two cysteines are conserved in all ALV subgroups, including ALV-J.…”