Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli.

Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and [methyl-3H]methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis (R. W. Bernlohr, A. L. Saha, C. C. Young, B. R. Toth, and K. J. Golden, J. Bacteriol. 170:4113-4118, 1988; K. J. Golden and R. W. Bernlohr, Mol. Gen. Genet. 220:1-7, 1989) and are consistent with the proposal that methylation of this protein functions in nutrient sensing.     

Heat-induced accumulation of chloroplast protein synthesis elongation factor, EF-Tu, in winter wheat

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum). In this study, we assessed heat tolerance and determined the relative levels of EF-Tu in mature plants (at flowering stage) of 12 cultivars of winter wheat experiencing a 16-d-long heat treatment (36/30 degrees C, day/night temperature). In addition, we also investigated the expression of EF-Tu in young plants experiencing a short-term heat shock (4h at 43 degrees C). Heat tolerance was assessed by examining the stability of thylakoid membranes, measuring chlorophyll content, and assessing plant growth traits (shoot dry mass, plant height, tiller number, and ear number). In mature plants, relative levels of EF-Tu were determined after 7 d of heat stress. High temperature-induced accumulation of EF-Tu in mature plants of all cultivars, and a group of cultivars that showed greater accumulation of EF-Tu displayed better tolerance to heat stress. Young plants of all cultivars but one did not show significant increases in the relative levels of EF-Tu. The results of the study suggest that EF-Tu protein may play a role in heat tolerance in winter wheat.     

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.     

Methylation in vivo of elongation factor EF-Tu at lysine-56 decreases the rate of tRNA-dependent GTP hydrolysis

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II is. Although the apparent affinity of tRNA for site II remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed.     

In vitro methylation of the elongation factor EF-Tu from Escherichia coli

The in vitro methylation of the elongation factor EF-Tu from Escherichia coli was investigated. The methylation of newly synthesized EF-Tu was obtained using lambda rifd 18 DNA as template and S-adenosyl [methyl-3H]methionine as methyl donor. About 3 mol methyl residues were incorporated for every 10 mol EF-Tu synthesized. Analysis of the nature of the methyl-containing residues by protein hydrolysis followed by paper chromatography showed that both mono- and dimethyllysine were present. The methylation of EF-Tu was also studied separately from its synthesis by using cell-free systems with artificially undermethylated components.     

In vivo methylation of prokaryotic elongation factor Tu.

In Salmonella typhimurium and Escherichia coli, elongation factor Tu (EF-Tu) is methylated as shown by its incorporation of labeled methyl residues from [methyl-3H]methionine. Analysis of the nature of the methyl-containing residues by protein hydrolysis, followed by paper chromatography and high voltage electrophoresis showed that both mono- and dimethyllysine are present. Eighty per cent of the EF-Tu molecules are methylated if methylation occurs at a unique lysine residue. The EF-Tu fraction which is not methylated is still able to accept methyl groups, as shown by methylation of approximately 10% of the EF-Tu after addition of chloramphenicol (D-(-)-threo-2,2-dichloro-N-[beta-hydroxy-alpha-(hydroxymethyl)-o-nitrophenethyl] acetamide) to inhibit further protein synthesis. There is no evidence of turnover of the methyl residues. We attempted to separate the methylated from the nonmethylated form of EF-Tu by isoelectric focusing on polyacrylamide gel, but were unable to do so.     

In vivo methylation of elongation factor Tu of Escherichia coli.

A protein existing mainly in the supernatant fraction of Escherichia coli was found to be methylated by accepting the methyl moiety originating from methionine. The protein was identified as peptide synthesis elongation factor Tu (EF-Tu) by the following criteria. 1) The methylatable protein separated at the same position as purified EF-Tu on two-dimensional gel electrophoresis. 2) The methylatable protein interacted with antiserum specific for EF-Tu. Amino acid analysis of the methyl-labeled protein suggested that the site of methylation was an epsilon-amino group of lysine.     

Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.     

Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.     

The elongation factor Tu from Escherichia coli, aminoacyl-tRNA, and guanosine tetraphosphate form a ternary complex which is bound by programmed ribosomes

The interaction of the Escherichia coli elongation factor Tu guanosine tetraphosphate complex (EF-Tu ppGpp) with aminoacyl-tRNAs(aa-tRNA) was reinvestigated by gel filtration and hydrolysis protection experiments. These experiments show that EF-Tu X ppGpp like EF-Tu X GDP (Pingoud, A., Block, W., Wittinghofer, A., Wolf, H. & Fischer, E. (1982) J. Biol. Chem. 257, 11261-11267) forms a fairly stable complex with Phe-tRNAPhe, KAss being 0.6 X 10(5) M-1 at 25 degrees C. The binding of the EF-Tu X ppGpp X aa-tRNA complex to programmed ribosomes was investigated by a centrifugation technique. It is shown that this complex is bound codon-specific with KAss = 3 X 10(7) M-1 at 0 degrees C and that it stimulates peptidyl transfer. A numerical estimation of the intracellular concentration of EF-Tu X GTP X aa-tRNA and EF-Tu X ppGpp X aa-tRNA during normal growth and under the stringent response indicates that ppGpp accumulation does affect the EF-Tu X GTP X aa-tRNA concentration but does not lead to major depletion of this pool. Furthermore, due to the higher affinity of EF-Tu X GTP to aa-tRNA and of the ternary complex EF-Tu X GTP X aa-tRNA to the ribosome, EF-Tu X ppGpp X aa-tRNA binding to the ribosome is not significant. According to our measurements and calculations, therefore, a direct participation of EF-Tu in slowing down the rate of protein biosynthesis and improving its accuracy during amino acid starvation is not obvious.     

A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria

Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.     

Altered regulation of the guanosine 5'-triphosphate activity in a kirromycin-resistant elongation factor Tu

In the preceding article a mutant elongation factor Tu (EF-TuD2216) resistant to the action of kirromycin was found to display a spontaneous guanosine 5'-triphosphatase (GTPase) activity, i.e., in the absence of aminoacyl transfer ribonucleic acid (tRNA) and ribosome-messenger RNA. This is the first example of an Ef-Tu supporting GTPase activity in the absence of macromolecular effectors and/or kirromycin. In this study we show that this activity is elicited by increasing NH4+ concentrations. As additional effect, the mutation caused an increased affinity of EF-Tu for GTP. Ammonium dependence of the GTPase activity an increased affinity for GTP are two properties also found with wild-type EF-Tu in the presence of kirromycin [Fasano, O., Burns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565; Sander, G., Okonek, M., Crechet, J.-B., Ivell, R., Bocchini, V., & Parmeggiani, A. (1979) FEBS Lett. 98, 111-114]. Therefore, both binding of kirromycin to wild-type EF-Tu and acquisition of kirromycin resistance introduce functionally related modifications. Kirromycin at high concentrations (0.1 mM) does not interact with mutant EF-TuD2216.GDP but still does with EF-TuD2216.GTP in agreement with our previous finding that EF-Tu.GTP is the preferential target of the antibiotic in the wild type [Fasano, O., Bruns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565). The GTPase activity of mutant EF-Tu in the presence of aminoacyl-tRNA and ribosome.mRNA is much higher than with wild-type EF-Tu and also much less dependent on the presence of mRNA. Miscoding for leucine, measured as poly(U)-directed poly(phenyl-alanine/leucine) synthesis at increasing Mg2+ concentrations, is identical for both wild-type and mutant EF-Tu.     

Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain

The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

An epitope of elongation factor Tu is widely distributed within the bacterial and archaeal domains.

A monoclonal antibody (MAb), MAb 900, which detects a 43-kDa protein present on Escherichia coli was found. Subsequently, more than 90 organisms, belonging to either the bacterial, archaeal, or eucaryal domain, were tested for reactivity to this MAb. Of the bacterial and archaeal domains, almost all species proved to be positive, whereas all organisms from the eucaryal domain gave negative results. The 43-kDa protein was purified by affinity chromatography and subsequently analyzed by microsequencing methods. Two peptide sequences which showed a high degree of homology (> 99%) to the prokaryotic elongation factor Tu (EF-Tu) were obtained. Western blot (immunoblot) analysis using both purified EF-Tu and EF-Tu domains confirmed that the unknown protein was EF-Tu. The panbacterial distribution of EF-Tu, which is present in large amounts in every prokaryotic cell, renders this protein a good candidate for a diagnostic approach. In consequence, we have used the anti-EF-Tu MAb 900 to design both a dot blot assay and an enzyme-linked immunosorbent assay. From either blood culture, urine, or gall-bladder fluid, bacterial contamination could be detected. The sensitivity of these tests is currently 10(4) bacteria per ml.     

Identification of the residues involved in the unique serine specificity of Caenorhabditis elegans mitochondrial EF-Tu2

In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.     

Histidine 66 in Escherichia coli elongation factor tu selectively stabilizes aminoacyl-tRNAs

The universally conserved His-66 of elongation factor Tu (EF-Tu) stacks on the side chain of the esterified Phe of Phe-tRNA(Phe). The affinities of eight aminoacyl-tRNAs were differentially destabilized by the introduction of the H66A mutation into Escherichia coli EF-Tu, whereas Ala-tRNA(Ala) and Gly-tRNA(Gly) were unaffected. The H66F and H66W proteins each show a different pattern of binding of 10 different aminoacyl-tRNAs, clearly showing that this position is critical in establishing the specificity of EF-Tu for different esterified amino acids. However, the H66A mutation does not greatly affect the ability of the ternary complex to bind ribosomes, hydrolyze GTP, or form dipeptide, suggesting that this residue does not directly participate in ribosomal decoding. Selective mutation of His-66 may improve the ability of certain unnatural amino acids to be incorporated by the ribosome.     

Structural characterization of novel complex oligosaccharides accumulated in the caprine beta-mannosidosis kidney. Occurrence of tetra- and pentasaccharides containing a beta-linked mannose residue at the nonreducing terminus

Four oligosaccharide fractions were isolated and purified from the kidney of goats affected with beta-mannosidosis by repeating Bio-Gel P-2 column chromatography. The structural characterization of the purified oligosaccharide fractions (oligosaccharides A, B, C1,2, and D) included sugar composition analysis by gas chromatography, sugar sequence analysis by mass spectrometry of their permethylated alditols, and by methylation analysis as well as anomeric configuration studies by exoglycosidase digestions. Oligosaccharides A and B were the major oligosaccharides accumulating in the kidney and were elucidated as Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc, respectively (Matsuura, F., Laine, R. A., and Jones, M. Z. (1981) Arch. Biochem. Biophys. 211, 485-493). Oligosaccharide C1,2 was a mixture of two tetrasaccharides and oligosaccharide D was a pentasaccharide. The proposed structures are: oligosaccharide C1, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc; oligosaccharide C2, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc; oligosaccharide D, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. Tetrasaccharide C1 and pentasaccharide D are heretofore undiscovered oligosaccharides. There is no precedent for these structures in glycoproteins or other glycoconjugates. One possibility which accounts for the presence of oligosaccharide C1 and D is that a bisecting N-acetylglucosamine (the beta-N-acetylglucosamine residue linked at the C-4 position of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chains) is linked by a beta-mannosyl residue. Moreover, the detection of oligosaccharides containing two N-acetylglucosamine residues at the reducing terminus, together with those containing a single N-acetylglucosamine residue, is further corroboration of species-specific differences in glycoprotein catabolic pathways (Hancock, L. W., and Dawson, G. (1984) Fed. Proc. 43, 1552) or in glycoprotein structures.