Specific in vitro O-glycosylation of human granulocyte-macrophage colony-stimulating-factor-derived peptides by O-glycosyltransferases of yeast and rat liver cells.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is O-glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS-1 cells [Ernst, J.F., Mermod, J.-J. and Richman, L.I. (1992) Eur. J. Biochem. 203, 663-667]. Two types of octapeptides encompassing residues 4-11 (peptide 4-11) and variants thereof, or residues 8-15 (peptide 8-15) of hGM-CSF were tested as substrates for in vitro O-glycosylation using dolichyl-phosphate- D-mannose: protein O-D-mannosyltransferase (Man-transferase) of the yeast Saccharomyces cerevisiae, or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) of rat liver cells. Peptide 8-15 was found to be O-glycosylated at residues Ser9 and Thr10 by GalNAc-transferase and, with reduced efficiency, also by Man-transferase. Peptide 4-11 was a good substrate for yeast Man-transferase, leading to mannosylation of only Thr10, whereas it was very poorly O-glycosylated at positions Ser5 and Ser7 by GalNAc-transferase. The observed differences in peptide-acceptor activities indicate that the site of O-glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.     

A sequence-coupled vector-projection model for predicting the specificity of GalNAc-transferase.

The specificity of GalNAc-transferase is consistent with the existence of an extended site composed of nine subsites, denoted by R4, R3, R2, R1, R0, R1', R2', R3', and R4', where the acceptor at R0 is either Ser or Thr to which the reducing monosaccharide is being anchored. To predict whether a peptide will react with the enzyme to form a Ser- or Thr-conjugated glycopeptide, a new method has been proposed based on the vector-projection approach as well as the sequence-coupled principle. By incorporating the sequence-coupled effect among the subsites, the interaction mechanism among subsites during glycosylation can be reflected and, by using the vector projection approach, arbitrary assignment for insufficient experimental data can be avoided. The very high ratio of correct predictions versus total predictions for the data in both the training and the testing sets indicates that the method is self-consistent and efficient. It provides a rapid means for predicting O-glycosylation and designing effective inhibitors of GalNAc-transferase, which might be useful for targeting drugs to specific sites in the body and for enzyme replacement therapy for the treatment of genetic disorders.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.     

Serine-324 of myosin's heavy chain is photoaffinity-labeled by 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate

A portion of the active site of rabbit skeletal myosin near the ribose ring of ATP can be labeled by the photoaffinity analogue 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate (Bz2ATP). The specificity of the photolabeling was assured by first trapping [14C]Bz2ATP at the active site by use of thiol cross-linking agents [Mahmood, R., Cremo, C., Nakamaye, K., & Yount, R. (1987) J. Biol. Chem. 262, 14479-14486]. Five radioactive peptides were isolated by high-performance liquid chromatography after extensive trypsin and subtilisin digestion of photolabeled myosin subfragment 1. Four of these peptides were sequenced by Edman techniques, and all originated from a region with the sequence Gly-Glu-Ile-Thr-Val-Pro-Ser-Ile-Asp-Asp-Gln, which corresponds to rabbit myosin heavy chain residues 318-328. The fifth labeled peptide had an amino acid composition appropriate for residues 312-328. Amino acid composition, radiochemical analysis, and sequence data indicate that Ser-324 is the major amino acid residue photolabeled by Bz2ATP. Spectrophotometric evidence indicates that the benzophenone carbonyl group has inserted into a C-H bond from either the alpha- or beta-carbon of serine. These results place Ser-324 at a distance of 6-7 A from the 3'(2') ribose oxygens of ATP bound at the active site of myosin.     

Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5'-diphosphate-alpha-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferases T1 and T2: kinetic modeling of the porcine and canine submaxillary gland mucin tandem repeats

A large family of uridine 5'-diphosphate (UDP)-alpha-N-acetylgalactosamine (GalNAc):polypeptide N-acetylgalactosaminyl transferases (ppGalNAc Ts) initiates mucin-type O-glycan biosynthesis at serine and threonine. The peptide substrate specificities of individual family members are not well characterized or understood, leaving an inability to rationally predict or comprehend sites of O-glycosylation. Recently, a kinetic modeling approach demonstrated neighboring residue glycosylation as a major factor modulating the O-glycosylation of the porcine submaxillary gland mucin 81 residue tandem repeat by ppGalNAc T1 and T2 [Gerken et al. (2002) J. Biol. Chem. 277, 49850-49862]. To confirm the general applicability of this model and its parameters, the ppGalNAc T1 and T2 glycosylation kinetics of the 80+ residue tandem repeat from the canine submaxillary gland mucin was obtained and characterized. To reproduce the glycosylation patterns of both mucins (comprising 50+ serine/threonine residues), specific effects of neighboring peptide sequence, in addition to the previously described effects of neighboring residue glycosylation, were required of the model. Differences in specificity of the two transferases were defined by their sensitivities to neighboring proline and nonglycosylated hydroxyamino acid residues, from which a ppGalNAc T2 motif was identified. Importantly, the model can approximate the previously reported ppGalNAc T2 glycosylation kinetics of the IgA1 hinge domain peptide [Iwasaki, et al. (2003) J. Biol. Chem. 278, 5613-5621], further validating both the approach and the ppGalNAc T2 positional weighting parameters. The characterization of ppGalNAc transferase specificity by this approach may prove useful for the search for isoform-specific substrates, the creation of isoform-specific inhibitors, and the prediction of mucin-type O-glycosylation sites.     

Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.     

The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the second largest cyanogen bromide fragment

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequence of the largest fragment, CNBr Ia has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequence of the 12 smaller fragments is reported in an accompanying paper (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1985) J. Biol. Chem. 261, 10511-10519). We have determined the amino acid sequence of the second largest fragment, CNBr Ib. The fragment contains 142 residues and has a molecular weight of 16,095.     

Predicting N-terminal acetylation based on feature selection method

Methionine aminopeptidase and N-terminal acetyltransferase are two enzymes that contribute most to the N-terminal acetylation, which has long been recognized as a frequent and important kind of co-translational modifications [R.A. Bradshaw, W.W. Brickey, K.W. Walker, N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families, Trends Biochem. Sci. 23 (1998) 263-267]. The combined action of these two enzymes leads to two types of N-terminal acetylated proteins that are with/without the initiator methionine after the N-terminal acetylation. To accurately predict these two types of N-terminal acetylation, a new method based on feature selection has been developed. 1047 N-terminal acetylated and non-acetylated decapeptides retrieved from Swiss-Prot database (http://cn.expasy.org) are encoded into feature vectors by amino acid properties collected in Amino Acid Index database (http://www.genome.jp/aaindex). The Maximum Relevance Minimum Redundancy method (mRMR) combining with Incremental Feature Selection (IFS) and Feature Forward Selection (FFS) is then applied to extract informative features. Nearest Neighbor Algorithm (NNA) is used to build prediction models. Tested by Jackknife Cross-Validation, the correct rate of predictors reach 91.34% and 75.49% for each type, which are both better than that of 84.41% and 62.99% acquired by using motif methods [S. Huang, R.C. Elliott, P.S. Liu, R.K. Koduri, J.L. Weickmann, J.H. Lee, L.C. Blair, P. Ghosh-Dastidar, R.A. Bradshaw, K.M. Bryan, et al., Specificity of cotranslational amino-terminal processing of proteins in yeast, Biochemistry 26 (1987) 8242-8246; R. Yamada, R.A. Bradshaw, Rat liver polysome N alpha-acetyltransferase: substrate specificity, Biochemistry 30 (1991) 1017-1021]. Furthermore, the analysis of the informative features indicates that at least six downstream residues might have effect on the rules that guide the N-terminal acetylation, besides the penultimate residue. The software is available upon request.     

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR study of molecular structure of the heme cavity

The molecular structure of the active site of myoglobin from the shark, Galeorhinus japonicus, has been studied by 1H-NMR. Some hyperfine-shifted amino acid proton resonances in the met-cyano form of G. japonicus myoglobin have been unambiguously assigned by the combined use of various two-dimensional NMR techniques; they were compared with the corresponding resonances in Physter catodon myoglobin. The orientations of ThrE10 and IleFG5 residues relative to the heme in G. japonicus met-cyano myoglobin were semiquantitatively estimated from the analysis of their shifts using the magnetic susceptibility tensor determined by a method called MATDUHM (magnetic anisotropy tensor determination utilizing heme methyls) [Yamamoto, Y., Nanai, N. & Ch?j?, R. (1990) J. Chem. Soc., Chem. Commun., 1556-1557] and the results were compared with the crystal structure of P. catodon carbonmonoxy myoglobin [Hanson, J. C. & Schoenborn, B. P. (1981) J. Mol. Biol. 153, 117-124]. In spite of a substantial difference in shift between the corresponding amino acid proton resonances for the two proteins, the orientations of these amino acid residues relative to the heme in the active site of both myoglobins were found to be highly alike.     

Rat liver NAD(P)H:quinone reductase: isolation of a quinone reductase structural gene and prediction of the NH2 terminal sequence of the protein by double-stranded sequencing of exons 1 and 2

Recently two reports [J. A. Robertson et al. (1986) J. Biol. Chem. 261, 15794-15799 and R. M. Bayney et al. (1987) J. Biol. Chem. 262, 572-575] have appeared concerning the nucleotide sequence of quinone reductase cDNA clones. Although the cDNA clones are virtually identical, they diverge in the 5' region that encodes the NH2 terminus of the protein. In order to clarify the sequence of this region, we have isolated quinone reductase clones from a rat genomic library using a cDNA clone, pDTD55, isolated and characterized by our laboratory. We have determined the sequence of exons 1 and 2 of the structural gene by double-stranded sequencing using oligonucleotide primers. The sequence of exons 1 and 2 of the quinone reductase structural gene along with our previous nucleotide sequence analysis of pDTD55 as well as conventional amino acid sequence analysis of the purified protein indicates that quinone reductase is composed of 274 amino acids with a molecular weight of 30,946. These data agree with the published sequence of lambda NMOR1 reported by Robertson et al.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Amino acid sequence of a calcium-binding protein (TCBP-10) from Tetrahymena

The amino acid sequence of a calcium-binding protein obtained from the cilium and cell body of Tetrahymena, designated as TCBP-10 (Tetrahymena calcium-binding protein; molecular mass = 10 kDa [Ohnishi, K. and Watanabe, Y. (1983) J. Biol. Chem. 258, 13978-13985] was determined. It is composed of 102 amino acid residues. The exact molecular mass is calculated to be 11563 Da. From the amino acid sequence analysis, it has two EF-hand-type calcium-binding sites.     

Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli.

The DNA sequence of a 3.23-kilobase fragment of the Escherichia coli chromosome encoding biosynthetic arginine decarboxylase (ADC) was determined. This sequence contained the speA open reading frame (ORF) as well as partial speB and metK ORFs. The ADC ORF is 1,974 nucleotides long; the deduced polypeptide contains 658 amino acids with a molecular size of 73,980 daltons. The molecular weight and predicted ADC amino acid composition are nearly identical to the amino acid analysis of purified ADC performed by Wu and Morris (J. Biol. Chem. 248:1687-1695, 1973). A translational speA-lacZ fusion, pRM65, including 1,389 base pairs (463 amino acids) of the 5' end of speA was constructed. Western blots (immunoblots) with beta-galactosidase antisera revealed two ADC::beta-galactosidase fusion proteins in E. coli bearing pRM65: 160,000 and 156,000 daltons representing precursor and mature hybrid proteins, respectively. The predicted amino acid sequence of ADC contains a region of six amino acid residues found in two bacterial diaminopimelic acid decarboxylases and three eucaryotic ornithine decarboxylases. This conserved sequence is located approximately eight amino acids from the putative pyridoxal phosphate-binding site of ADC and is predicted to be involved in substrate binding.     

Structural determinants within residues 180-199 of the rodent alpha 5 nicotinic acetylcholine receptor subunit involved in alpha-bungarotoxin binding

Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) alpha subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. We have previously defined alpha-bungarotoxin (alpha-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR alpha subunit, designated alpha 5 [McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 9816-9824], and between residues 181 and 200 of the chick neuronal alpha 7 and alpha 8 subunits [McLane, K. E., Wu, X., Schoepfer, R., Lindstrom, J., & Conti-Tronconi, B. M. (1991) J. Biol. Chem. (in press)]. These sequences are relatively divergent compared with the Torpedo and muscle nAChR alpha 1 alpha-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the alpha 5 sequence, we were interested in determining the critical amino acid residues for alpha-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat alpha 5(180-199) sequence were tested, using a competition assay, in which peptides compete for 125I-alpha-BTX binding with native Torpedo nAChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and molecular cloning of succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex. Absence of a sequence motif of the putative E3 and/or E1 binding site

Full-length cDNA clones for succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex were isolated from rat heart cDNA libraries in lambda gt11. The cDNA clones were identified as those for rat succinyltransferase by the identity of their predicted amino acid sequence with the NH2-terminal amino acid sequence of rat succinyltransferase determined by protein chemical analysis and the known amino acid sequence of bovine succinyltransferase. The clone with the longest cDNA consisted of 2747 base pairs and coded for a leader peptide of 56 amino acid residues and a mature protein of 386 amino acid residues. The primary structure of rat succinyltransferase showed close similarity to Escherichia coli and Azotobacter vinelandii succinyltransferases, in the COOH-terminal part forming the lipoyl-binding domain and the NH2-terminal part forming the inner core-catalytic domain. However, the rat succinyltransferase did not contain a sequence motif that has been found as an E3- and/or E1-binding site in the dihydrolipoamide acyltransferases of three alpha-ketoacid dehydrogenase complexes (Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J., and Yeaman, S. J. (1988) J. Biol. Chem. 263, 6165-6168, Reed, L. J., and Hackert, M. L. (1990) J. Biol. Chem. 265, 8971-8974). The absence of this sequence was confirmed by direct sequencing of the polymerase chain reaction product of rat heart mRNA and by computer analysis. These results show that the rat succinyltransferase does not have the sequence motif of the putative E3- and/or E1-binding site.     

Structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. Sequences of the overlapping peptides, ordering the CNBr fragments, and the complete amino acid sequence

The amino acid sequence of the largest fragment, CNBr Ia (203 residues) has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequences of the second largest fragment, CNBr Ib (142 residues), and of the 12 smaller fragments are reported in accompanying papers (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10511-10519; Behrens, P. Q., Nakashima, H., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). The complete amino acid sequence of hemocyanin component II has been established by isolation and analysis of 13 methionine-containing peptides from either a tryptic digest or a Staphylococcus aureus strain V8 protease digest of whole carboxamidomethylated hemocyanin II. Hemocyanin II is composed of 628 residues and has a molecular weight with two copper atoms of 72,946.     

The primary structure of skeletal muscle myosin heavy chain: II. Sequence of the 50 kDa fragment of subfragment-1

The complete amino acid sequence of the 50 kDa fragment of subfragment-1 from adult chicken pectoralis muscle myosin was determined. It contained 431 residues including an epsilon-N-trimethyllysine at position 346. The 431-residue sequence corresponds to the sequence of residues 206 to 639 of chicken embryonic breast muscle myosin heavy chain which was predicted from the nucleotide sequence of the cDNA by Molina et al. [Molina, M. I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488]. Comparing the two sequences, 23 amino acid substitutions and three deletions/insertions are recognized.     

Location of seven post-translational modifications in rabbit elongation factor 1 alpha including dimethyllysine, trimethyllysine, and glycerylphosphorylethanolamine

Amino acid sequencing of a large number of chemical and enzymatic cleavage products of elongation factor 1 alpha purified from rabbit reticulocyte has identified seven post-translationally modified residues. Five of the modifications are methylations of lysine residues yielding dimethyllysine at residues 55 and 165 and trimethyllysine at residues 36, 79, and 318. The two remaining post-translational modifications involve the addition of ethanolamine to glutamic acid residues 301 and 374, as reported previously (Rosenberry, T. L., Krall, J. A., Dever, T. E., Haas, R., Louvard, D., and Merrick, W. C. (1989) J. Biol. Chem. 264, 7096-7099). Fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry have been used to analyze peptides containing these modified residues. The analyses have determined that glycerylphosphorylethanolamine has been attached to the glutamic acid residues. An analysis of the amino acid sequence surrounding each of the three types of modification has indicated no similarities. Therefore, it seems likely that the modifying enzymes do not recognize a specific amino acid sequence but rather the three-dimensional presentation of either amino or carboxyl residues in the elongation factor 1 alpha structure.     

The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.