Phillip M. Strike

The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an α-chain that is entirely extracellular and a β-chain that spans the cell membrane. The α-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the β-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give α-β monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the α-α dimer bond Cys524. In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the β-chain residues Cys798 and Cys807 and between the α-chain Cys647 and the β-chain Cys872. The latter bond is the sole α-β link in the molecule and implies a side-by-side alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional α-α dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys884 exists as a buried thiol in the soluble ectodomain.

N-linked glycans of the human insulin receptor and their distribution over the crystal structure

The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N‐linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N‐linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N‐linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO‐K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR‐B numbering); multiple species of high‐mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high‐mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high‐mannose glycan species. Of the remaining five predicted N‐linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high‐mannose glycans lie at positions of relatively low steric accessibility. Proteins 2008.

Coat protein of potyviruses. 6. Amino acid sequences suggest watermelon mosaic virus 2 and soybean mosaic virus-N are strains of the same potyvirus.

SummaryThe amino acid sequence of the coat protein of watermelon mosaic virus 2 (WMV 2) was determined by a combination of peptide and nucleic acid sequencing. The coat protein of WMV 2 contained 281 amino acid residues including a single cysteine at position 132 and a blocked amino terminus. Comparison with the coat protein sequences of 20 strains of ten distinct potyviruses showed sequence homologies ranging from 43% to 69% except for the N strain of soybean mosaic virus (SMV-N), where the sequence homology with WMV 2 was 83%. This degree of homology and the location of sequence differences between WMV 2 and SMV-N is much closer to that observed between strains of the same virus than that found between distinct potyviruses. These data suggest that WMV 2 and SMV-N may be strains of the same virus.

Cowpea aphid borne mosaic virus - Morocco and South African Passiflora virus are strains of the same potyvirus.

SummaryHigh performance liquid chromatography (HPLC) profiles of tryptic peptides and partial amino acid sequence analysis have been employed to establish the taxonomic status of the Moroccan isolate of cowpea aphid-borne mosaic virus (CABMV). Some previous reports have suggested CABMV to be very closely related to blackeye cowpea mosaic virus (B1CMV) while other reports have concluded that this relationship is distant. In this report a tryptic digest of the coat protein of CABMV-Morocco was compared with those of the coat proteins of B1CMV-Type, B1CMV-W, the mild mottle strain of peanut stripe virus (PStV-MM) and the NY15 strain of bean common mosaic virus (BCMV-NY15), all of which are now recognised as strains of BCMV. The comparisons also included the NL-3 strain of bean necrosis mosaic virus (BNMV-NL3), which had previously been classified as a strain of BCMV. The HPLC peptide profiles indicated that CABMV-Morocco was distinct from BCMV and BNMV. Amino acid sequence analysis of peptides accounting for more than half of the coat protein confirmed that CABMV-Morocco was not a strain of BNMV or BCMV but was a distinct member of the BCMV subset of viruses that previously has been shown to include BCMV, BNMV, soybean mosaic virus, zucchini yellow mosaic virus, passionfruit woodiness virus and South AfricanPassiflora virus (SAPV). Comparison of the partial sequence data with these and other published sequences revealed that the coat protein of CABMV-Morocco is very similar to that of SAPV suggesting that they are strains of the same virus. Since CABMV was described over 25 years earlier than SAPV, the name CABMV should take precedence and SAPV should be renamed CABMV-SAP, the South AfricanPassiflora strain of CABMV.

The location and characterisation of the O‐linked glycans of the human insulin receptor

O‐linked glycosylation is a post‐translational and post‐folding event involving exposed S/T residues at β‐turns or in regions with extended conformation. O‐linked sites are difficult to predict from sequence analyses compared to N‐linked sites. Here we compare the results of chemical analyses of isolated glycopeptides with the prediction using the neural network prediction method NetOGlyc3.1, a procedure that has been reported to correctly predict 76% of O‐glycosylated residues in proteins. Using the heavily glycosylated human insulin receptor as the test protein six sites of mucin‐type O‐glycosylation were found at residues T744, T749, S757, S758, T759, and T763 compared to the three sites (T759 and T763‐ correctly, T756‐ incorrectly) predicted by the neural network method. These six sites occur in a 20 residue segment that begins nine residues downstream from the start of the insulin receptor β‐chain. This region which also includes N‐linked glycosylation sites at N742 and N755, is predicted to lack secondary structure and is followed by residues 765–770, the known linear epitope for the monoclonal antibody 18–44. Proteins 2007.

Coat protein of potyviruses 7. Amino acid sequence of peanut stripe virus.

SummaryThe amino acid sequence of the 287-residue coat protein of peanut stripe virus (PStV) was determined from the sequences of overlapping peptide fragments. Results indicated that the amino terminus was blocked by an acetyl group, as has previously been found for the coat protein of Johnsongrass mosaic potyvirus. Comparison of the PStV sequence with coat proteins of 20 distinct potyviruses gave sequence identities of 47–57%, except for zucchini yellow mosaic virus (ZYMV), passionfruit woodiness virus (PWV), and the related strains watermelon mosaic virus 2 (WMV 2) and soybean mosaic virus-N, which showed sequence identities of 70–76%. Several amino acid residues which were common to the core sequences of these coat proteins were at positions previously found to be invariant among potyvirus coat proteins. The degree of these similarities suggests that although PStV, WMV 2, ZYMV, and PWV are distinct potyviruses, they share a common ancestor in their evolutionary development.

Watermelon mosaic virus-Morocco is a distinct potyvirus

SummaryThe relationship of the Morocco isolate of watermelon mosaic virus (WMV) to WMV 2, soybean mosaic virus (a virus closely related to WMV 2) and the W strain of papaya ringspot virus (PRSV-W), formerly WMV 1, was examined by comparing tryptic peptide profiles using high performance liquid chromatography. The profiles indicated that the coat protein sequence of WMV-Morocco differed substantially from those of the other potyviruses. This conclusion was supported by sequence data from five tryptic peptides from the coat protein of WMV-Morocco, which showed only 61–68% identity to equivalent sequences in PRSV-W, WMV 2 and zucchini yellow mosaic, another potyvirus infecting cucurbits. Based on the above data, and on known correlations between coat protein sequence similarities and potyvirus relationship, it is concluded that WMV-Morocco should be regarded as a distinct potyvirus.

Amino acid sequence of the acidic kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.

Amino acid sequences of pilins from serologically distinct strains of Bacteroides nodosus

Amino acid sequences of pilin from a strain of Bacteroides nodosus from serogroup B (234) and serogroup C (217) were determined. The amino-terminal N-methylphenlalanine residue of both proteins was followed by a hydrophobic sequence of 30 residues closely related to the N-terminal sequence of other pili having an amino-terminal residue of N-methylphenylalanine. These data lend support to the hypothesis that in pilins of this type, the amino-terminal sequence functions as a transport signal necessary for pilin to reach its external environment, as well as promoting intersubunit interactions for muintenance of the structural integrity of the pilus. Two hydrophilic hypervariable regions can be discerned across the pilin sequences, indicating possible locations of antigenic domains.

Primary structure of kunitz-type trypsin inhibitor-2a (pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a,pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a,pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2,pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1,pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.