Sex effect on the correlation of immunoglobulin G glycosylation with rheumatoid arthritis disease activity.

Rheumatoid arthritis (RA) is a chronic autoimmune disease which affects females more than males with a presence of autoantibodies. Immunoglobulin G (IgG) produced by adaptive arm has 2 functional domains, Fc and Fab. The Fc domain binds Fc gamma receptors and C1q proteins of the innate arm. Therefore, the IgG Fc domain serves as a bridge between the innate and adaptive arms and is regulated by an evolutionarily conserved N-glycosylation with variable structures. These glycans are classified as agalactosylated G0, monogalactosylated G1, and digalactosylated G2, which are further modified by core-fucosylation (F) and bisecting N-acetylglucosamine (B) moieties such as G0F and G0FB. Interestingly, proinflammatory G0F is shown to be regulated by estrogen in vivo. Here, it is hypothesized that the regulation of G0F by estrogen contributes to sex dichotomy in RA by setting up the level of IgG-dependent inflammation and therefore, RA disease activity (Das28-CRP3). To investigate this hypothesis, IgG glycosylation was characterized in serum samples from active RA patients (n = 232) and healthy controls (n = 232) by serum N-glycan analysis using the high performance liquid chromatography. According to the results, the IgG Fc glycan phenotype originates predominantly from the structure of G0F, and both G0F and G0FB correlate with Das28-CRP3 in females, but not in males. In conclusion, IgG G0F-dependent inflammation differs in males and females, and these differences point to the differential regulation of inflammation by sex hormone estrogen via IgG glycosylation.

High-throughput characterization of the functional impact of IgG Fc glycan aberrancy in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation.

Estrogens regulate glycosylation of IgG in women and men.

The immunologic potency of IgG is modulated by glycosylation, but mechanisms regulating this process are undefined. A role for sex hormones is suggested by differences in IgG glycans between women and men, most prominently with respect to galactose. We therefore assessed IgG galactosylation in 713 healthy adults from 2 cohorts as well as in 159 subjects from 4 randomized controlled studies of endocrine manipulation: postmenopausal women receiving conjugated estrogens, raloxifene, or placebo; premenopausal women deprived of gonadal hormones with leuprolide and treated with estradiol or placebo; men deprived of gonadal hormones with goserelin and given testosterone or placebo; and men deprived of gonadal hormones with goserelin and given testosterone or placebo together with anastrozole to block conversion of testosterone to estradiol. Menopause was associated with an increase in agalactosylated IgG glycans, particularly in the most abundant fucosylated nonbisected (G0F) glycoform. Conjugated estrogens and raloxifene reduced G0F glycans in postmenopausal women, while in premenopausal women leuprolide increased G0F glycans in a manner reversed by estradiol. Among men, goserelin increased G0F glycans, an effect blocked by testosterone through conversion to estradiol. These results establish estrogens as an in vivo modulator of IgG galactosylation in both women and men, defining a pathway by which sex modulates immunity.

Expression patterns of bovine CD1 in vivo and assessment of the specificities of the anti-bovine CD1 antibodies.

Research addressing the in vivo effects of T cell activation by lipids, glycolipids, and lipopeptides is hampered by the absence of a suitable animal model. Mice and rats do not express CD1a, CD1b, and CD1c molecules that present pathogen-derived lipid antigens in humans. In cattle, two CD1A and three CD1B genes are transcribed. The proteins encoded by these genes differ in their antigen binding domains and in their cytoplasmic tails, suggesting that they may traffic differently in the cell and thus have access to different antigens. In the current study, we describe the genomic organization of the bovine CD1 locus and transcription of bovine CD1 genes in freshly isolated dendritic cells and B cells from different tissues. After determining the specificity of previously only partly characterized anti-CD1 antibodies by testing recombinant single chain bovine CD1 proteins and CD1-transfected cells, we were able to determine cell surface protein expression on freshly isolated cells. Our study suggests that CD1b1 and CD1b3 are more broadly expressed than CD1b5, and CD1a2 is more broadly expressed than CD1a1. Pseudoafferent lymph dendritic cells express CD1B genes, but no transcription is detected in lymph nodes. Even though B cells transcribe CD1B genes, there is no evidence of protein expression at the cell surface. Thus, patterns of CD1 protein expression are largely conserved among species.

Congenital disorder of fucosylation type 2c (LADII) presenting with short stature and developmental delay with minimal adhesion defect.

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migration caused by mutations in the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.

Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity.

While the induction of a neutralizing antibody response against HIV remains a daunting goal, data from both natural infection and vaccine-induced immune responses suggest that it may be possible to induce antibodies with enhanced Fc effector activity and improved antiviral control via vaccination. However, the specific features of naturally induced HIV-specific antibodies that allow for the potent recruitment of antiviral activity and the means by which these functions are regulated are poorly defined. Because antibody effector functions are critically dependent on antibody Fc domain glycosylation, we aimed to define the natural glycoforms associated with robust Fc-mediated antiviral activity. We demonstrate that spontaneous control of HIV and improved antiviral activity are associated with a dramatic shift in the global antibody-glycosylation profile toward agalactosylated glycoforms. HIV-specific antibodies exhibited an even greater frequency of agalactosylated, afucosylated, and asialylated glycans. These glycoforms were associated with enhanced Fc-mediated reduction of viral replication and enhanced Fc receptor binding and were consistent with transcriptional profiling of glycosyltransferases in peripheral B cells. These data suggest that B cell programs tune antibody glycosylation actively in an antigen-specific manner, potentially contributing to antiviral control during HIV infection.

Characterization of fibrinogen glycosylation and its importance for serum/plasma N-glycome analysis.

The majority of proteins present in human serum/plasma are glycoproteins, validating this fluid as an ideal starting material for N-glycan analysis and discovery of potential biomarkers. The glycoprotein content for both serum and plasma is very similar, except for proteins removed in the coagulation process, including fibrinogen. Our aim was to characterize fibrinogen glycosylation in order to determine its contribution to differences between serum and plasma N-glycomes. N-Glycans from human fibrinogen were released, labeled, and analyzed by HILIC-HPLC and MS. Structural characterization of fibrinogen subunits revealed that the α chain was not N-glycosylated, whereas ß and γ contained identical oligosaccharide structures, mainly biantennary digalactosylated monosialylated structures (A2G2S1) and biantennary digalactosylated disialylated structures (A2G2S2). Blood was collected from five healthy volunteers into four testing tubes: silicone-coated glass for serum and EDTA, Na-heparin, and Li-heparin glass tubes for plasma. N-Glycans were analyzed using the high-throughput HILIC-HPLC method. N-Glycan profiles from serum and plasma samples differed largely in glycans identified in fibrinogen, suggesting that this glycoprotein represents a major factor distinguishing these body fluids. This result emphasizes the important of consistent body fluid collection practices in biomarker discovery studies.

Multiple juvenile idiopathic arthritis subtypes demonstrate proinflammatory IgG glycosylation.

OBJECTIVE: Rheumatoid arthritis is associated with an excess of agalactosylated (G0) IgG that is considered relatively proinflammatory. Assessment of this association in juvenile idiopathic arthritis (JIA) is complicated by age-dependent IgG glycan variation. The aim of this study was to conduct the first large-scale survey of IgG glycans in healthy children and patients with JIA, with a focus on early childhood, the time of peak JIA incidence. METHODS: IgG glycans from healthy children and disease-modifying antirheumatic drug-naive patients with JIA were characterized using high-performance liquid chromatography. Agalactosylated glycans were quantitated with reference to monogalactosylated (G1) species. Associations were sought between the G0:G1 ratio and disease characteristics. RESULTS: Among healthy children ages 9 months to 16 years (n = 165), the G0:G1 ratio was highly age dependent, with the ratio peaking to 1.19 in children younger than age 3 years and declining to a nadir of 0.83 after age 10 years (Spearman's ρ = 0.60, P < 0.0001). In patients with JIA (n = 141), the G0:G1 ratio was elevated compared with that in control subjects (1.32 versus 1.02; P < 0.0001). The G0:G1 ratio corrected for age was abnormally high in all JIA subtypes (enthesitis-related arthritis was not assessed), most strikingly in systemic JIA. Glycosylation aberrancy was comparable in patients with and those without antinuclear antibodies and in both early- and late-onset disease and exhibited at most a weak correlation with markers of inflammation. CONCLUSION: IgG glycosylation is skewed toward proinflammatory G0 variants in healthy children, in particular during the first few years of life. This deviation is exaggerated in patients with JIA. The role for IgG glycan variation in immune function in children, including the predilection of JIA for early childhood, remains to be defined.

Hypogalactosylation of serum N-glycans fails to predict clinical response to methotrexate and TNF inhibition in rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is associated with hypogalactosylation of immunoglobulin G (IgG). We examined whether a proxy measure for galactosylation of IgG N-glycans could predict response to therapy or was differentially affected by methotrexate (MTX) or TNF blockade. METHODS: Using a previously defined normal phase high-performance liquid chromatography approach, we ascertained the galactosylation status of whole serum N-glycans in two well-defined RA clinical cohorts: the Autoimmune Biomarkers Collaborative Network (n = 98) and Nested I (n = 64). The ratio of agalactosylated to monogalactosylated N-glycans in serum (sG0/G1) was determined before and during therapy with MTX or TNF inhibition and correlated with anticitrullinated peptide antibody (ACPA) status and clinical response as assessed by 28-joint Disease Activity Score utilizing C-reactive peptide and European League Against Rheumatism response criteria. RESULTS: RA patients from both cohorts exhibited elevation of sG0/G1 at baseline. Improvement in clinical scores correlated with a reduction in sG0/G1 (Spearman's ρ = 0.31 to 0.37; P < 0.05 for each cohort). However, pretreatment sG0/G1 was not predictive of clinical response. Changes in sG0/G1 were similar in the MTX and TNF inhibitor groups. Corrected for disease activity, ACPA positivity correlated with higher sG0/G1. CONCLUSIONS: Baseline serum N-glycan hypogalactosylation, an index previously correlated with hypogalactosylation of IgG N-glycans, did not distinguish patients with rheumatoid arthritis who were likely to experience a favorable clinical response to MTX or TNF blockade. Clinical improvement was associated with partial glycan normalization. ACPA-positive patients demonstrated enhanced N-glycan aberrancy compared with ACPA-negative patients.

Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis.

OBJECTIVE: To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA). METHODS: Analysis of N-glycan in serum samples from multiple cohorts was performed. The IgG N-glycan content and the timing of N-glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anti-cyclic citrullinated peptide (anti-CCP) antibody titer, disease duration, and C-reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N-glycan content was also compared between epitope affinity-purified autoantibodies and the remaining IgG repertoire in RA patients. RESULTS: Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean +/- SD 1.36 +/- 0.43 versus 1.01 +/- 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's rho = 0.37, P < 0.0001). This correlation was higher in women (Spearman's rho = 0.60, P < 0.0001) than in men (Spearman's rho = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction. CONCLUSION: Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex-specific manner, and resides preferentially in autoantibodies.

Mechanistic role of each metal ion in Streptomyces dinuclear aminopeptidase: PEPTIDE hydrolysis and 7x10(10)-fold rate enhancement of phosphodiester hydrolysis.

The dinuclear aminopeptidase from Streptomyces griseus (SgAP) and its metal derivatives catalyze the hydrolysis of the phosphoester bis(p-nitrophenyl) phosphate (BNPP) and the phosphonate ester p-nitrophenyl phenylphosphonate with extraordinary rate enhancements at pH 7.0 and 25 degrees C [A. Ercan, H. I. Park, L.-J. Ming, Biochemistry 45, (2006) 13779-13793.], reaching 6.7 billion-fold in terms of the first-order rate constant of the di-Co(II) derivative with respect to the autohydrolytic rates. Since phosphoesters are transition state-like inhibitors in peptide hydrolysis, their hydrolysis by SgAP is quite novel. Herein, we report the investigation of this proficient alternative catalysis of SgAP and the role of each metal ion in the dinuclear site toward peptide and BNPP hydrolysis. Mn(II) selectively binds to one of the dinuclear metal sites (M1), affording MnE-SgAP with an empty (E) second site for the binding of another metal (M2), including Mn(II), Co(II), Ni(II), Zn(II), and Cd(II). Peptide hydrolysis is controlled by M2, wherein the k(cat) values for the derivatives MnM2-SgAP are different yet similar between MnCo- and CoCo-SgAP and pairs of other metal derivatives. On the other hand, BNPP hydrolysis is affected by metals in both sites. Thus, the two hydrolytic catalyses must follow different mechanisms. Based on crystal structures, docking, and the results presented herein, the M1 site is close to the hydrophobic specific site and the M2 site is next to Tyr246 that is H-bonded to a coordinated nucleophilic water molecule in peptide hydrolysis; whereas a coordinated water molecule on M1 becomes available as the nucleophile in phosphodiester hydrolysis.

A "moonlighting" dizinc aminopeptidase from Streptomyces griseus: mechanisms for peptide hydrolysis and the 4 x 10(10)-fold acceleration of the alternative phosphodiester hydrolysis.

A unique "enzyme catalytic promiscuity" has recently been observed, wherein a phosphodiester and a phosphonate ester are hydrolyzed by a dinuclear aminopeptidase and its metal derivatives from Streptomyces griseus (SgAP) [Park, H. I., Ming, L.-J. (1999) Angew. Chem., Int. Ed. Engl. 38, 2914-2916 and Ercan, A., Park, H. I., Ming, L.-J. (2000) Chem. Commun. 2501-2502]. Because tetrahedral phosphocenters often serve as transition-state inhibitors toward the hydrolysis of the peptide, phosphoester hydrolysis by peptidases is thus not expected to occur effectively and must take place through a unique mechanism. Owing to the very different structures and mechanistic requirements between phosphoesters and peptides during hydrolysis, the study of this effective phosphodiester hydrolysis by SgAP may provide further insight into the action of this enzyme that is otherwise not obtainable from regular peptide substrates. We present herein a detailed investigation of both peptide and phosphodiester hydrolyses catalyzed by SgAP. The latter exhibits a first-order rate enhancement of 4 x 10(10)-fold compared to the uncatalyzed reaction at pH 7.0 and 25 degrees C. The results suggest that peptide and phosphodiester hydrolyses by SgAP may share a common reaction mechanism to a certain extent. However, their differences in pH dependence, phosphate and fluoride inhibition patterns, and proton inventory reflect that they must follow different pathways. Mechanisms for the two hydrolyses are drawn on the basis of the results, which provide the foundation for further investigation of the catalytic promiscuity of this enzyme by means of physical and molecular biology methods. The catalytic versatility of SgAP suggests that this enzyme may serve as a unique "natural model system" for further investigation of dinuclear hydrolysis. A better understanding of enzyme catalytic promiscuity is also expected to shed light on the evolution and action of enzymes.

Molecular characterization of a novel UDP-galactose:fucoside alpha3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.

The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-alpha class of animal prolyl 4-hydroxylases.

Skp1 is a cytoplasmic and nuclear protein of eukaryotes best known as an adaptor in SCF ubiquitin-protein isopeptide ligases. In Dictyostelium, Skp1 is subject to 4-hydroxylation at Pro(143) and subsequent O-glycosylation by alpha-linked GlcNAc and other sugars. Soluble cytosolic extracts have Skp1 prolyl 4-hydroxylase (P4H) activity, which can be measured based on hydroxylation-dependent transfer of [(3)H]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline alpha-N-acetyl-d-glucosaminyltransferase. The Dictyostelium Skp1 P4H gene (phyA) was predicted using a bioinformatics approach, and the expected enzyme activity was confirmed by expression of phyA cDNA in Escherichia coli. The purified recombinant enzyme (P4H1) was dependent on physiological concentrations of O(2), alpha-ketoglutarate, and ascorbate and was inhibited by CoCl(2), 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIFalpha) class. Overexpression of phyA cDNA in Dictyostelium yielded increased enzyme activity in a soluble cytosolic extract. Disruption of the phyA locus by homologous recombination resulted in loss of detectable activity in extracts and blocked hydroxylation-dependent glycosylation of Skp1 based on molecular weight analysis by SDS-PAGE, demonstrating a requirement for P4H1 in vivo. The sequence and functional similarities of P4H1 to animal HIFalpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIFalpha, be regulated by availability of O(2), alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.

Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase from Dictyostelium.

Mucin-type O-glycosylation in Dictyostelium is initiated in the Golgi by a UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase (Dd-pp alphaGlcNAcT2) whose sequence is distantly related to the sequences of animal polypeptide-Thr/Ser N-acetyl-alpha-galactosaminyltransferases, such as murine Mm-pp alphaGalNAcT1. To evaluate the significance of this similarity, highly purified Dd-pp alphaGlcNAcT2 was assayed using synthetic peptides derived from known substrates. Dd-pp alphaGlcNAcT2 strongly prefers UDP-GlcNAc over UDP-GalNAc, preferentially modifies the central region of the peptide, and modifies Ser in addition to Thr residues. Initial velocity measurements performed over a matrix of UDP-GlcNAc donor and peptide acceptor concentrations indicate that the substrates bind to the enzyme in ordered fashion before the chemical conversion. Substrate inhibition exerted by a second peptide, and the pattern of product inhibition exerted by UDP, suggest that UDP-GlcNAc binds first and the peptide binds second, consistent with data reported for Mm-pp alphaGalNAcT1. Two selective competitive inhibitors of Mm-pp alphaGalNAcT1, retrieved from a screen of neutral-charge uridine derivatives, also inhibit Dd-pp alphaGlcNAcT1 competitively with only slightly less efficacy. Inhibition is specific for Dd-pp alphaGlcNAcT2 relative to two other Dictyostelium retaining glycosyltransferases. These data support a phylogenetic model in which the alphaGlcNAcT function in unicellular eukaryotes converted to an alphaGalNAcT function in the metazoan ortholog while conserving a similar reaction mechanism and active site architecture.

Specificity of a soluble UDP-galactose: fucoside alpha1,3-galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium.

Skp1 is an adaptor-like protein in E3(SCF)-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galalpha1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn(2+) and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K(m) and 35-fold higher V(max) compared with a simple alpha-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V(max) advantage, but still much less active than Skp1 itself because of a K(m) difference. These findings indicate that alpha-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

Proteolytic susceptibility of creatine kinase isozymes and arginine kinase.

The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.