Immunochemical studies on the cross-reactivity between streptococcal and staphylococcal mucopeptide.

Particulate mucopeptides of Group A-variant streptococci and Staphylococcus aureus, solubilized by ultrasonic treatment, give a precipitin reaction with the sera of rabbits immunized with Group A-variant streptococci. gamma-G globulin antibodies have been recovered from these sera which react with the mucopeptides but not with the Group A-variant carbohydrate. The immunochemical basis for the cross-reactivity between the streptococcal and staphylococcal mucopeptides was investigated in detail. Three chemically different fractions have been isolated from enzymatic digests of staphylococcal mucopeptide and were employed as haptenic inhibitors of the precipitin reaction. A fraction consisting of the peptide moiety of mucopeptide was the strongest inhibitor, whereas the hexosamine-rich fraction was less effective. The third fraction, rich in glycine, was least effective. It is suggested that the immunologic cross-reactivity between streptococcal and staphylococcal mucopeptide is due to the fact that these two substances contain chemically similar tetrapeptides. The hexosamine polymer which is identical for both mucopeptides may also contribute to their cross-reactivity.

Induction of rabbit antibody with molecular uniformity after immunization with group C streptococci.

Antibodies with uniform properties may occur in rabbits after immunization with Group C streptococci. These precipitating antibodies possess specificity for the group-specific carbohydrate. Not uncommonly, their concentration is between 20 and 40 mg/ml of antiserum. Evidence for molecular uniformity in the case of one of these antibodies, described in detail here, includes: individual antigenic specificity; monodisperse distribution of the light chains by alkaline urea polyacrylamide disc electrophoresis; and a single amino acid in each of the first three N-terminal positions of the light chains. When the amino acid sequence of rabbit antibody b+ light chains (kappa type) are aligned against their human kappa counterparts, a definite homology is observed between the N-terminus of the human and the rabbit variable region.

New approaches for the laboratory recognition of M types of group A streptococci.

The successful classification of Group A streptococci by the capillary precipitin technique requires a complete series of M type antisera which are sufficiently potent and specific to give unequivocal type-specific reactions with all the serotypes. Specific antisera for this purpose have been prepared by absorption with heterologous streptococci. Unabsorbed antisera have been employed here in the Ouchterlony double-diffusion agar-gel test to identify the M type of streptococci. Techniques have been developed for making this method of M typing fully reliable. The results reported here confirm and amplify the original findings of Michael and Massell (3). With crude HCl extracts and unabsorbed M type antisera, a precipitin line due to the M protein and another to the group-specific carbohydrate are the two major reactions observed. These reactions, however, are usually readily distinguishable. There was a surprising lack of cross-reactive precipitin lines due to non-type-specific protein antigens in the extracts. Although many of the unabsorbed M type antisera can be employed in the double-diffusion tests, the group-specific antibody must be removed from some of the unabsorbed antisera to avoid confusing cross-reactions. Absorption of these antibodies has been achieved by means of a specific immunoabsorbent column prepared from para-aminophenyl-beta-N-acetylglucosamine and cyanogen bromide-activated Sepharose. Excellent agreement was observed between the M typing results obtained on 117 field strains by the conventional capillary precipitin method and the Ouchterlony double-diffusion method.

Identification of HE1 as the second gene of Niemann-Pick C disease.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

Molecular mimicry in Lyme disease: monoclonal antibody H9724 to B. burgdorferi flagellin specifically detects chaperonin-HSP60.

A monoclonal antibody (H9724), specific for the 41-kDa flagellar protein of the Lyme disease pathogen Borrelia burgdorferi, cross-reacts with human axons and detects one major protein in human neuroblastoma cell extracts. The homologous cross-reacting protein has now been isolated from calf adrenal and identified as chaperonin-HSP60 by N-terminal sequencing.

An acridine amino acid derivative for use in Fmoc peptide synthesis.

An acridine derivative of N-alpha-Fmoc-lysine has been prepared and used in solid phase peptide synthesis. The fluorescence properties of the acridine reporter group are retained throughout the peptide synthesis procedure. The utility of the acridine group was demonstrated by its use as an energy acceptor in a fluorescence energy-quenching assay with trypsin.

The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH.

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.

Proteolytic cleavage of subunits of the nucleocapsid of the paramyxovirus simian virus 5.

The protein subunits of the nucleocapsid of the parainfluenza virus simian virus 5 isolated from infected cells after dispersion with trypsin, chymotrypsin, or ficin are cleaved proteolytically. The molecular weights of the subunits which result from cleavage depend on the enzyme used, but are around 43,000, compared to the native subunit of 61,000. In most instances cleavage of the subunit appears to be due to the protease used to disperse the cell, and follows cell disruption. Nucleocapsids composed of native, uncleaved subunits can frequently be obtained from infected cells dispersed without a proteolytic enzyme; however, cleavage occasionally occurs even under those conditions, indicating that cellular proteases can at times cleave this protein. Nucleocapsids containing uncleaved subunits can be isolated from cells persistently infected with simian virus 5, indicating that persistent infection is not invariably associated with intracellular cleavage of this protein. Nucleocapsids composed of native subunits are hydrophobic, whereas those composed of the cleaved subunit can be dispersed in aqueous solution. It is suggested that the portion of the molecule removed by cleavage may be responsible for a specific interaction during virus assembly between the nucleocapsid and those areas of plasma membrane which contain the non-glycosylated viral membrane protein, which is also hydrophobic. An amino acid analysis of native and cleaved subunits has been done. The portion of the subunit removed by cleavage does not have a high proportion of hydrophobic residues, suggesting that those present are arranged together to form a hydrophobic domain. The N termini of both the native and cleaved subunits are blocked. This suggests that the portion of the molecule which is externally disposed and removed by cleavage contains the C terminus, and the cleaved subunit which reacts with the viral RNA contains the N terminus.

Covalent attachment of peptides to membranes for dot-blot analysis of glycosylation sites and epitopes.

Noncovalent binding of proteins to membranes is often employed for dot-blot analysis with various visualization techniques. These techniques are usually not applicable to peptide dot-blot analysis due to peptide wash-off during the staining procedure. As exemplified with a synthetic peptide and peptides produced by proteolysis of a protein, it is possible to achieve efficient covalent attachment to Immobilon-AV membranes. The utility of this membrane has been demonstrated with immunostaining and carbohydrate staining procedures.

Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio.

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.

Mannose 6-phosphate (Man-6-P) is a posttranslational carbohydrate modification typical of newly synthesized acid hydrolases that signals targeting from the Golgi apparatus to the lysosome via Man-6-P receptors (MPRs). Using iodinated cation independent MPR as a probe in a Western blot assay, we surveyed levels of Man-6-P glycoproteins in a number of different rat tissues. Considerable variation was observed with respect to total amounts and types of Man-6-P glycoproteins in the different tissues. Brain contained 2-8-fold more Man-6-P glycoproteins than other tissues, with relative abundance being brain >> testis approximately heart > lung approximately kidney approximately ovary approximately spleen > skeletal muscle approximately liver approximately serum. Analysis of 16 different lysosomal enzyme activities revealed that brain contains lower activities than other tissues which suggested that decreased removal of Man-6-P results in increased levels of Man-6-P glycoproteins. This was directly demonstrated by comparing activities of phosphorylated lysosomal enzymes, purified by immobilized MPR affinity chromatography, with total activities. The phosphorylated forms accounted for a considerable proportion of the MPR-targeted activities measured in brain (on average, 36.2%) but very little in lung, kidney, and liver (on average, 5.5, 2.3, and 0. 7%, respectively). Man-6-P glycoproteins were also isolated from rat brain by MPR affinity chromatography on a preparative scale. Of the 18 bands resolvable by SDS-polyacrylamide gel electrophoresis, seven bands were NH2-terminally sequenced and identified as the known lysosomal enzymes cathepsin L, cathepsin A, cathepsin D, alpha-galactosidase A, arylsulfatase A, and alpha-iduronidase. One of the major Man-6-P glycoproteins was identified as palmitoyl protein thioesterase, which was not previously thought to be lysosomal. This finding raises important questions about the cellular location and function of palmitoyl protein thioesterase, mutations in which result in the neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis.

The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A.

Human platelet arylsulphatase A (ASA) exhibits a multiple banding pattern when examined by PAGE. The isoform pattern (IVa) of the enzyme obtained from normal subjects differs from variants (IIIa, IIIb, IVb) which are primarily found in alcoholic patients. Alkaline phosphatase and endo-N-acetylglucosaminidase H treatments, as well as ion-exchange chromatography, demonstrate that the isoforms of ASA arise because of charge heterogeneity caused by phosphoglycan moieties. The isoform with the slowest mobility in PAGE lacks phosphate substituents. Based upon mannose-6-phosphate-receptor affinity chromatography it can be concluded that most, if not all, of the enzyme-linked phosphate is in the form of 6-O-phospho-D-mannosyl units. Lectin affinity chromatography and peptide-N-glycosidase F treatment followed by SDS/PAGE and Western-blot analysis indicate that normal platelet ASA contains two oligomannose and/or hybrid glycan moieties of which one, or both, possess a 6-O-L-fucosyl substituent on the asparagine-linked N-acetylglucosamine residue. Comparative analysis indicates that the variant IIIa and IIIb types of ASA differ from the IVa ASA with regard to the level of glycan phosphorylation and glycan structure, as well as the polypeptide structure.

alpha-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine.

Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and alpha-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were beta-mannosidase (82%), hexosaminidase (27%) and alpha-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.

Chemical studies on the structure of mucopeptide isolated from Streptococcus bovis.

Mucopeptides isolated from Streptococcus bovis cell walls were found to be composed of alanine, glutamic acid, lysine, and threonine in a mole ratio of 3:1:1:1. A dipeptide, N(epsilon)-lysylthreonine, was isolated from S. bovis mucopeptide by ion-exchange chromatography. This finding suggests that threonine is associated with the bridge which cross-links adjacent tetrapeptides by connecting the epsilon-amino group of lysine of one tetrapeptide to the carboxyl group of d-alanine of another to form the mucopeptide matrix.

Presence of an N-terminal polyhistidine tag facilitates stable expression of an otherwise unstable N-terminal domain of mouse tissue inhibitor of metalloproteinase-1 in Escherichia coli.

The active N-terminal domain of the mouse tissue inhibitor of metalloproteinases-1 is a 14.1-kDa polypeptide with three disulfide bonds. When expressed using a T7 system in Escherichia coli, this truncated protein, in contrast to the WT protein, was found only in trace amounts in the cell. However, when the coding sequence was placed downstream of a 60-bp sequence that encoded an in-frame histidine-rich "tag," the fusion product (NF.TIMP*His) was expressed in considerably increased abundance. WT.TIMP-1 was expressed in abundance with or without the tag. The mRNAs encoding the various forms of TIMP were present in similar amounts in all four cases. NF.TIMP*His, renatured and purified on a nickel affinity column, was found to be about 10-fold less effective than native human TIMP-2 at inhibiting cleavage of collagen type I by human fibroblast collagenase. A thrombin cleavage site in the tag was susceptible to cleavage by low levels of a contaminating proteinase.

Analysis of the conformational stability of the active domain of recombinant mouse TIMP-1 by intrinsic fluorescence.

Intrinsic fluorescence was used to examine the stability of an active, N-terminal domain of mouse tissue inhibitor of metalloproteinase (TIMP-1) fused with an N-terminal polyhistidine tag. Emission and quenching studies suggested that the single tryptophan is on the protein surface partially exposed to solvent. The TIMP-1 recombinant unfolded reversibly in the presence of guanidinium chloride with the transition midpoint at 2.35M; extrapolation gave a stabilization free energy of 5.1 kcal mol-1 at 25 degrees C. Analysis of the temperature dependence of the fluorescence intensity gave a melting transition with midpoint at 51 degrees C and an enthalpy and heat capacity change on unfolding of 32 kcal mol-1 and 0.45 kcal K-1 mol-1, respectively, values comparable to other single domain proteins. Comparison with literature data indicated that the stability of mouse recombinant TIMP-1 more closely resembled that of human metalloproteinase inhibitor TIMP-2 than TIMP-1 despite closer homology to the human TIMP-1 protein.

Amino acid analysis on polyvinylidene difluoride membranes.

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.

Presentation of peptide antigens as albumin conjugates for use in detection of serum antibodies by enzyme-linked immunosorbent assay.

The use of linear peptides as antigens for detection of serum antibodies has been studied using a sequence of the Borrelia burgdorferi protein, flagellin, and Lyme disease sera as a model. It was found that a novel presentation of the peptide as a hapten on the carrier protein, bovine serum albumin, in the enzyme-linked immunosorbent assay format can be successfully applied to distinguish between Lyme disease and control sera.

Amino acid sequence restriction in rabbit antibody light chains.

Light chains were obtained from IgG rabbit antibodies to the group-specific carbohydrates of groups A and C streptococci. An analysis of the amino acid alternatives which exist at the first three positions of the N-terminus in both light-chain preparations shows a marked restriction in amino acid sequence heterogeneity when compared with preimmune light chains. Both of these related, but immunologically distinct, antigenic determinants select the same uncommon subpopulation of rabbit light chains.