Tryptophan glycoconjugates in food and human urine

Properdin is the positive regulator of the alternative pathway of complement activation. The 53-kDa protein is essentially composed of six thrombospondin type 1 repeats, all of which contain the WXXW motif, the recognition sequence for C-mannosylation. C-Mannosylation is a post-translational modification of tryptophan residues in which, in contrast to the well known N-and O-glycosylation, the carbohydrate is attached via a C-C bond to C-2 of the indole moiety of tryptophan. C-Mannosylation was first found in human RNase 2 and interleukin-12. The terminal complement proteins C6 -C9 also carry this modification as part of their thrombospondin type 1 repeats. We studied the C-mannosylation pattern of human properdin by mass spectrometry and Edman degradation. Properdin contains 20 tryptophans of which 17 are part of a WXXW motif. Fourteen tryptophans were found to be modified 100%. This is the first example of a protein in which the majority of tryptophan residues occurs in the C-mannosylated form. These results show that C-mannosylated proteins occur at several steps along the complement activation cascade. Therefore, this system would be ideal to investigate the function of C-mannosylation.Glycosylation is one of the most abundant and widespread post-translational modifications of proteins. In the common cases of N-and O-glycosylation the glycan is attached via an amide or a hydroxyl group of an amino acid side chain to the protein. In human RNase 2 a fundamentally different type of glycosylation was discovered (1-3). An ␣-mannosyl residue was found to be linked via a C-C bond to the C-2 of the indole ring of tryptophan (Fig. 1). It was shown that this modification is enzyme-catalyzed and that dolichylphosphate mannose is the sugar donor (4). In RNase 2 the recognition signal for the transferase was determined to be WXXW (or less efficiently WXXF), in which the first tryptophan becomes modified (5). Meanwhile a total of 22 tryptophans in 7 proteins have been shown to be C-mannosylated, i.e. RNase 2 (1), interleukin-12 (6), and terminal complement proteins C6, C7, C8␣ and ␤, and C9 (7). The terminal complement proteins showed a more complex pattern of C-mannosylated tryptophans than RNase 2 and interleukin-12. They contain as a part of their thrombospondin repeats (TSRs) 1 WXXWXXW motifs, in which both of the first two tryptophans and, surprisingly, also the last one can be C-mannosylated. In addition, in TSRs of C6 and C7, tryptophans were found to be modified although they were not even part of a WXXW motif (7).The complement system is an innate first line host defense mechanism against microbes. Its activation pathways are composed of three proteolytic cascades, which merge at the cleavage step of (inactive) C3, converting it into active C3b ( Fig. 2; reviewed in Ref. 8). Properdin (or factor P) is a positive regulator of the complement system. It stabilizes the C3 convertase (C3bBb) in the feedback loop of the alternative pathway (Fig. 2), protecting it from rapid inactivation (9 -11). In addition, the C5 con...

Section: Fig 4 Esimsms Of the [M ؉ 2h]supporting

confidence: 73%

Properdin, the Positive Regulator of Complement, Is HighlyC-Mannosylated

Hartmann

Hofsteenge

2000

“…S4), suggesting a cleavage that is typical of O-and N-linked hexoses (20,21). The 367-Da ion and its fragmentation, most notably the 120-Da loss to m/z 247, are identical with standard C 2 -[␣-D-mannopyranosyl]-tryptophan (22,23) (Fig. 4).…”

Section: Resultsmentioning

confidence: 61%

“…It was first reported in pancreatic ribonuclease and since then in over 47 other proteins including notably thrombospondin and complement proteins with the sequence motif WX 1 X 2 W (20,(22)(23)(24)(25)(26). Although the function of this modification remains elusive, mannosylation is known to render the tryptophan more polar and solvent-accessible (23).…”

mentioning

confidence: 99%

Glycosylated Hydroxytryptophan in a Mussel Adhesive Protein from Perna viridis

Zhao

Sagert

Hwang

et al. 2009

The 3,4-dihydroxyphenyl-L-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW 1 TAW 2 K and APPPAW 1 TAW 2 K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C 2 -hexosylated tryptophan (W 1 ) and C 2 -hexosylated hydroxytryptophan (W 2 ), the latter of which is redox active. The UV absorbance spectrum of W 2 is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion.The amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) 3 occurs in many proteins of the mussel holdfast or byssus (1, 2) and has recently been incorporated into mussel-inspired synthetic polymers with versatile adhesive consequences (3-7). One byssal protein in particular, mussel foot protein-1 (Mfp-1), has been investigated from over 15 mussel byssi, where it protectively coats compliant collagen-like proteins in the thread core (8 -11). Mfp-1s typically contain 10 -15 mol % Dopa in a highly conserved repeating peptide structure (8). In the blue mussel Mytilus edulis fp-1 (Mefp-1), for example, the consensus decapeptide AKPSYPPTYK is repeated over 70 times in tandem, and much of the tyrosine (Y) is converted to Dopa (Y*) (Fig. 1). In stark contrast to this, only trace levels of Dopa were detected in Pvfp-1 from the green mussel Perna viridis (Linnaeus 1758) (12), a notoriously invasive fouling species originally from the Indo-Pacific region (13). Because P. viridis fp-1 (Pvfp-1) and its homologue, Mefp-1, are both strongly aromatic, quinogenic, and composed of highly polar decapeptide repeats (12), identifying the Dopa-mimetic substitutes in Pvfp-1 has been a matter of considerable interest. MATERIALS AND METHODSProtein Isolation from Mussel Feet-Green mussels (P. viridis) were collected from Tampa harbor in Florida. The feet were severed from about 100 freshly shucked mussels and stored at Ϫ80°C. Protein extraction from mussel feet was adapted from a previous report (12). Frozen mussel feet (in lots of 10 g of wet weight) were thawed and depigmented by scraping with a scalpel. The depigmented feet were homogenized in a glass tissue grinder with 50 ml of 5% acetic acid and two protease inhibitors (pepstatin A and leupeptin, both 30 mM). The homogenate was centrifuged (15,000 ϫ g, 4°C, 30 min), and the recovered supernatant (S1) was chilled in an ice bath. 70% perchloric acid was added dropwise with stirring to a final perchloric acid concentration of 1.4% (v/v). The mixtur...

“…whether this new type of protein glycosylation is common in living organisms and what is the biochemical mechanism leading to the Trp N-mannosylation in proteins. Formation of N-mannosyl Trp proceeds under high acidic conditions at elevated temperature (22); consequently, one may ask if the N-linked mannose might be produced during CPO isolation. All buffers used for CPO purification were near neutral pH, and most of the purification procedures were conducted at 4°C.…”

Section: Possibility Of Trp N-mannosylation As a Common Post-translatmentioning

confidence: 99%

“…Further analysis of their MS/MS spectra determined that the mannosyl moiety is not linked via the anomeric carbon to the C2 atom of the Trp indole ring as those described for a number of mammalian proteins (2-4) but is covalently connected via the N-1 atom of the side chain of Trp. Although a Trp N-linked glycoconjugate has been detected in fruit (21,22), N-mannosylation of the peptideassociated Trp has not been clearly identified as a protein post-translational event or as a new type of protein glycosylation. The detection of Trp N-mannosylation in CPO raises some interesting questions, such as questions concerning the enzymes involved in catalyzing the Trp N-mannosylation or hydrolyzing the Trp N-linked mannose in proteins and, more importantly, the physiological function of the Trp N-mannosylation in proteins, which should stimulate further research in these directions.…”

mentioning

confidence: 99%

Novel Glycosidic Linkage in Aedes aegypti Chorion Peroxidase

Rock

2005

Aedes aegypti chorion peroxidase (CPO) plays a crucial role in chorion hardening by catalyzing chorion protein cross-linking through dityrosine formation. The enzyme is extremely resistant to denaturing conditions, which seem intimately related to its posttranslational modifications, including disulfide bond formation and glycosylation. In this report, we have provided data that describe a new type of glycosylation in CPO, where a mannose is linked to the N-1 atom of the indole ring of Trp residue. Through liquid chromatography/electrospray ionization/tandem mass spectrometry and de novo sequencing of CPO tryptic peptides, we determined that three of the seven available Trp residues in mature CPO are partially (40 -50%) or completely mannosylated. This conclusion is based on the following properties of the electrospray ionization/tandem mass spectrometry spectra and the enzymatic reaction of these peptides Glycosylation is the most abundant post-translational modification in proteins. Based on the linkage of oligosaccharides with amino acid residues on proteins, O-glycosylation and N-glycosylation are the two best known types and have been extensively studied (1). In O-glycosylation, sugar is attached to the hydroxyl group of serine, threonine, tyrosine, or other amino acid residues through a hydroxyl group. In N-glycosylation, oligosaccharide is linked to the peptide through N-acetylglucosamine and asparagine with a recognition sequence of Asn-X-Ser/Thr, where X can be any amino acid except for proline. During the past 10 years, a new type of protein glycosylation involving attachment of an ␣-mannose to the C-2 carbon of the indole ring of Trp residues in proteins has been reported (2-4). It is termed C-glycosylation because this linkage is formed via a C-C bond. C-mannosylation has been proved to occur in a number of mammalian proteins, such as RNase2, interleukin-12, complements, properdin, thrombospondin, erythropoietin receptor, mucins, and a bovine lens protein (2-11, 13-16). A Trp-X-X-Trp (WXXW) motif seems to serve as the specificity determinant for C-mannosylation, in which the first Trp residue is mannosylated. In addition, it has been demonstrated that the C-mannosylation is an enzyme-catalyzed event (17). The potential function of the C-mannosylation has also been discussed in a recent report (15).In our study dealing with mosquito chorion hardening, we identified a specific chorion peroxidase (CPO) 2 that displayed extremely high physical stability under a number of denaturing conditions. For example, the enzyme remains active for months in 1-5% SDS (18).CPO undergoes extensive post-translational modifications, including proteolytic processing and glycosylation (19). Recently, we have determined the N-glycosylation site and N-glycan structures in CPO (20). In our current study dealing with the characterization of CPO post-translational modifications, we determined that some Trp residues in CPO were mannosylated. Further analysis of their MS/MS spectra determined that the mannosyl moiety is not lin...