Allen M. Gold

Trichinella spiralis: Secreted antigen of the infective L1 larva localizes to the cytoplasm and nucleoplasm of infected host cells

Antibodies were elicited against a purified antigen with an apparent molecular weight of 43K. This antibody preparation also detected a second antigen consisting of a group of closely related components of 45-50K. These antigens are stage specific for the infective first stage larva of Trichinella spiralis and are among the repertoire of secreted antigens originating from the stichosome. Antibody raised against the 43K antigen reacted with the stichosome and cuticle of the mature larva and the cytoplasm and nucleoplasm, but not nucleolus, of all nuclei of infected host cells (Nurse cells) in sections of infected tissues. Studies on sections of synchronously infected muscle tissue revealed that antigen was present only within the worm on Day 7 of the infection. On Day 9 after infection, the stichosome and cuticular surface of the larva and the cytoplasm and nucleoplasm of each nucleus of the Nurse cell reacted with antibody. Nurse cell cytoplasmic and nuclear reactivity increased in intensity until Day 18 after infection. These results suggest that stichocyte-specific antigens are synthesized during the early phase of infection in the muscle, and that as the Nurse-parasite complex develops, some of the antigen is secreted into the milieu of the Nurse cell. The presence of antigen in the cytoplasm and nucleoplasm of the infected host cell is discussed in relation to Nurse cell formation and maintenance.

Partial characterization of two antigens secreted by L1 larvae of Trichinella spiralis

Two protein antigens were isolated from excretory-secretory products of Trichinella spiralis by biochemical methods and characterized with respect to their chemical and immunological properties. One antigen, of apparent Mr 43,000, is an abundant secreted protein of infective L1 larvae, while the other, of 45-50 kDa, is present in smaller amounts. Yields, extinction coefficients, isoelectric points, amino acid compositions, and partial N-terminal amino acid sequences for each are reported. Partial amino acid sequences of peptides derived from the 43-kDa protein by cyanogen bromide cleavage have been determined. Treating a reduced-pyridylethylated derivative of the 43-kDa protein with glycopeptidase F (N-glycanase) resulted in formation of a transient product of 37 kDa followed by a stable polypeptide of 32 kDa (by SDS-PAGE), suggesting the presence of two N-linked carbohydrate groups. A similar result was obtained with the 45-50-kDa protein, which gave a transient doublet of 38 and 40 kDa and a final, stable product of 33 kDa, with a minor component of 35 kDa. Two glycosylation sites of the 43-kDa protein and one site of the 45-50-kDa protein can be identified in the amino acid sequences. Polyclonal antibodies prepared against the two proteins cross-reacted extensively, but failed to react with the doubly deglycosylated polypeptides in Western blots. The dominant epitopes present in the reduced-pyridylethylated polypeptides are, therefore, N-linked carbohydrate, although the presence of peptide epitopes in the native proteins cannot be excluded.

Developmental expression of a 43-kDa secreted glycoprotein from Trichinella spiralis

Trichinella spiralis is an intracellular parasitic nematode that infects skeletal muscle cells. Infection results in loss of tissue specific characteristics and conversion of the muscle cell to a Nurse cell. The characteristic changes leading to the formation of the Nurse cell appear complete by day 12 after intramuscular infection. Proteins synthesized in the stichocytes (secretory cells) of T. spiralis and secreted in the host cell are believed to be involved in the process of Nurse cell formation. One secreted glycoprotein of 43 kDa has been considered as a candidate factor involved in Nurse cell formation. We determined the timing of synthesis and secretion of the 43-kDa glycoprotein and its temporal correlation to the changes of the infected host cell, to gain an understanding of the role of the 43-kDa glycoprotein in T. spiralis infection. We show that the 43-kDa glycoprotein is first expressed on day 11 following intramuscular infection, several days after the changes in the infected muscle cell have been initiated. Protein(s) immunologically related to the 43-kDa glycoprotein but not the 43-kDa glycoprotein itself are detected in the nuclei of mature Nurse cells. During the intramuscular stage the 43-kDa glycoprotein appears to be stored in the alpha-stichocytes of T. spiralis and appears to be secreted immediately following invasion of the intestinal columnar epithelial cells by the L1 larva. The role of the 43-kDa glycoprotein remains unknown, however, these findings argue against involvement of the 43-kDa glycoprotein in Nurse cell formation.

α-D-Glucopyranosyl fluoride: A substrate of sucrose phosphorylase

α-D-Glucopyranosyl fluoride (glucosyl fluoride) was found to be a substrate for sucrose phosphorylase from Pseudomonas saccharophila. At saturating Pi (0.030 M) the apparent Km is the same as that of sucrose, but the apparent Vmax is 2.7 times as great; therefore, glucosyl fluoride is the best glucose donor presently known for sucrose phosphorylase. The enzyme catalyzes hydrolysis of glucosyl fluoride at the same rate as sucrose and glucose-1-P.

Glycogen branching enzyme in Lafora myoclonus epilepsy

Abstract Glycogen branching enzymes (EC 2.4.1.18) in extracts of cerebral cortex from a patient with Lafora myoclonus epilepsy and several normal subjects were compared in terms of activity and physical properties of the proteins. Branching-enzyme activity in Lafora cortex tissue was significantly higher than in controls. Gel-exclusion chromatography revealed a single branching-enzyme species of identical elution volume in normal and pathological tissues. A molecular weight of 95,000 was estimated for the enzyme from normal cerebral cortex by means of sucrose density gradient sedimentation. A specific staining method for detection of branching-enzyme activity in polyacrylamide gel slabs was developed for the analysis of electropherograms of tissue extracts. Normal cortex extracts showed the presence of two or more closely spaced bands of branching activity; Lafora cortex extracts showed four strongly stained bands which migrated more rapidly toward the anode than those from the normal tissue. We conclude that Lafora myoclonus epilepsy does not involve a deficiency of total branching-enzyme activity but the protein appears to be of a slightly more acidic nature than that of normal tissue. This may reflect alterations in another system involved in protein processing.

Trichinella pseudospiralis secretes a protein related to the Trichinella spiralis 43-kDa glycoprotein

A 43-kDa secreted glycoprotein from the intracellular parasitic nematode Trichinella spiralis has been considered as a factor involved in the formation of the Nurse cell in infected muscle. The closely related intracellular parasitic nematode Trichinella pseudospiralis that also infects muscle cells, does not form Nurse cells and was thought not to secrete the 43-kDa glycoprotein. This implied a unique role for the 43-kDa glycoprotein in T. spiralis infection and supported the hypothesis of involvement of the 43-kDa glycoprotein in Nurse cell formation. Following cloning of a full length cDNA encoding the 43-kDa protein, antibodies were raised against several domains of the 43-kDa glycoprotein. Here we show that a protein related to the 43-kDa glycoprotein exists in T. pseudospiralis. Immunohistochemical studies reveal important similarities in the distribution of the 43-kDa glycoprotein and the related protein from T. pseudospiralis in muscle infections with either of the two parasites. The 43-kDa glycoprotein may therefore play a common role in the life cycles of these two parasites and probably is not involved in Nurse cell formation.

On the mechanism of glycogen phosphorylase

Abstract The rate of exchange of 18 O from the COP position into the external positions of the phosphate group of glucose 1-phosphate was studied. An indirect method, involving acid-catalyzed exchange of 18 O into the 1-position of glucose 6-phosphate followed by isomerization with phosphoglucomutase, was used to prepare a rapidly equilibrating mixture in which glucose 1-phosphate with 18 O in the COP position was a component. This mixture was treated with phosphorylase a alone and with phosphorylase a and glycogen as a control to indicate complete exchange. Isotopic oxygen that had migrated to external positions of the phosphate group was estimated by treating the mixture of phosphates with alkaline phosphatase in ordinary water, isolating KH 2 PO 4 , and determining its isotope content. The rate of exchange catalyzed by phosphorylase a was no greater than 0.3% of the potential catalytic rate in the presence of saturating glycogen. It is concluded that no observable breakage of the CO bond of glucose 1-phosphate occurs in the binary complex with phosphorylase a .

Occurrence of high and low Mr forms of glycogen phosphorylase in extracts of human brain

Although glycogen phosphorylase (EC 2.4.1 .l) from rabbit muscle has been subjected to the most detailed analysis of structure, mechanism and function, other glycogen phosphorylases have received less attention. The human brain enzyme in particular, has been neglected in many respects. It is known that this tissue contains three isoenzymes which correspond to a predominant brain type, muscle type, and a hybrid of the two [1,2]. Here, we have investigated the characteristics of human brain phosphorylase by means of gel-exclusion chromatography and sucrose density gradient sedimentation. Both phosphorylase a and b appear to exist in a highMr form of -400 000 and a low Mr form whose apparent size differs with the method of determination. Although the nature of the high MI form is not known, we believe that it reflects a previously unknown property of human brain phosphorylase. The low M, form is probably an equilibrium mixture of dimer and monomer which gives different apparent Mr-values depending upon the position of equilibrium. For gel-exclusion chromatography the extract was treated as described in the text and a 1.5 ml aliquot containing 300 mg sucrose was layered on a 1.4 cm X 94 cm column of Sephacryl S-300 (Pharmacia, Uppsala) equilibrated with a buffer containing 0.20 M Tris-HCl (PH 7.2) 0.20 M NaCl, and 5 mM 2-mercaptoethanol. The column was eluted with the same buffer at 5°C with a flow rate of 12 ml/h and fractions of 1.5 ml were collected. Sucrose density gradient sedimentation was done as in [3] with a SW 50.1 rotor, using 4.6 ml gradients of 5-20% sucrose containing 5 mM 2-mercaptoethanol and indicated concentrations of Tris-HCl (pH 7.2) and NaCl. The gradients were collected in 0.20 ml fractions. Phosphorylase was assayed as in [4] in a final volume of 50 fl; 2 mM AMP was included for the assay of phosphorylase b. Orthophosphate was determined as in [5].