Phosphohexosyl recognition is a general characteristic of pinocytosis of lysosomal glycosidases by human fibroblasts.

We recently presented data showing that mannose-6-phosphate was a potent competitive inhibitor of pinocytosis of human platelet beta-glucuronidase, and that treatment of "high-uptake" forms of the enzyme with alkaline phosphatase destroyed the high-uptake property of the enzyme without diminishing its catalytic activity. These data indicate that phosphate is a necessary component of the recognition marker on the enzyme for pinocytosis by human fibroblasts, and suggest that the phosphate on high-uptake forms of the enzyme is present as a phosphohexosyl moiety. Results presented here show that mannose-6-phosphate is also a potent inhibitor of pinocytosis of the following enzyme preparations: (a) beta-glucuronidase from human spleen, liver, placenta, and urine; (b) beta-hexosaminidase and beta-galactosidase from human platelets; (c) beta-hexosaminidase from human fibroblast secretions. Alkaline phosphatase treatment of all these enzymes except beta-galactosidase, which was unstable to the incubation conditions and could not be tested, greatly diminished the uptake activity of the enzymes without diminishing their catalytic activity. These results suggest that phosphohexosyl recognition is a general characteristic of pinocytosis of lysosomal glycosidases.

Altered mRNA metabolism in ribonuclease III-deficient strains of Escherichia coli.

The metabolism of mRNA from the lactose (lac) operon of Escherichia coli has been studied in ribonuclease (RNase) III-deficient strains (rnc-105). The induction lag for beta-galactosidase from the first gene was twice as long, and enzyme synthesis was reduced 10-fold in one such mutant compared with its isogenic rnc+ sister; in the original mutant strain AB301-105, synthesis of beta-galactosidase was not even detectable, although transduction analysis revealed the presence of a normal lac operon. This defect does not reflect a loss of all lac operon activity galactoside acetyltransferase from the last gene was synthesized even in strain AB301-105 but at a rate several times lower than normal. Hybridization analyses suggested that both the frequency of transcription initiation and the time to transcribe the entire operon are normal in rnc-105 strains. The long induction lag was caused by a longer translation time. This defect led to translational polarity with reduced amounts of distal mRNA to give a population of smaller-sized lac mRNA molecules. All these pleiotropic effects seem to result from RNase III deficiency, since it was possible to select revertants to rnc+ that grew and expressed the lac operon at normal rates. However, the rnc-105 isogenic strains (but not AB301-105) also changed very easily to give a more normal rate of beta-galactosidase synthesis without regaining RNase III activity or a faster growth rate. The basis for this reversion is not known; it may represent a "phenotypic suppression" rather than result from a stable genetic change. Such suppressor effects could account for earlier reports of a noninvolvement of RNase III in mRNA metabolism in deliberately selected lac+ rnc-105 strains. The ribosomes from rnc-105 strains were as competent as ribosomes from rnc+ strains to form translation initiation complexes in vitro. However, per mass, beta-galactosidase mRNA from AB301-105 was at least three times less competent to form initiation complexes than was A19 beta-galactosidase mRNA. RNase III may be important in the normal cell to prepare lac mRNA for translation initiation. A defect at this step could account for all the observed changes in lac expression. A potential target within a secondary structure at the start of the lac mRNA is considered. Expression of many operons may be affected by RNase III activity; gal and trp operon expressions were also abnormal in RNase III- strains.

Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.

Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is specifically taken up from the culture medium by human fibroblasts. Prior work has indicated that the enzyme exhibits charge heterogeneity and that "high-uptake" forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than slowly internalized forms. Here we present two lines of evidence that the acidic group required for the high-uptake property of certain forms of the enzyme is a phosphate on, or in proximity to, a D-mannose-type carbohydrate. The first line of evidence was obtained from analysis of inhibition of enzyme pinocytosis by yeast mannans, phosphorylated sugars, and sugars. Mannans that contained phosphate were more potent inhibitors than those that did not contain phosphate. D-Mannose 6-phosphate was a more potent inhibitor than either D-mannose 1 phosphate or 2-deoxy-D-glucose 6-phosphate. D-Mannose and certain related sugars were weak pinocytosis inhibitors, while 2- and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and the apparent Kis estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnnl, 3 X 10(-6) M; mannan from wild-type S. cerebisiae, 3 X 10(-5) M; D-mannose 6-phosphate, 6 X 10(-5) M; L-fucose, 4 X 10(-2) M; and D-mannose, 6 X 10(-2) M. The second line of evidence comes from the observation that alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] treatment of human platelet beta-glucuronidase abolished its "high-uptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.

Human beta-glucuronidase. II. Fate of infused human placental beta-glucuronidase in the rat.

Human placental beta-glucuronidase could be identified in rat organs by heating organ extracts to 65 degrees which selectively inactivated endogenous rat enzyme. Infused enzyme was rapidly cleared from rat plasma (t0.5 of 3.5 min). From 50-60% of the infused dose was accounted for in rat liver and spleen 24 hr after infusion. Removal of abdominal viscera, including the spleen, and interruption of the portal circulation before infusion slowed the plasma disappearance (t0.5 of 60 min) and allowed significant uptake by bone and other organs. Subcellular fractionation of liver 18 hr postinfusion localized the human enzyme in the mitochondrial-lysosomal fraction. The half-disappearance times of infused human enzyme were 2.6 days in rat liver and 5.8 days in rat spleen. Periodate treatment of human placental beta-glucuronidase destroyed 90% of its binding to concanavalin A-Sephadex rose, reduced its heat stability, and abolished its rapid clearance from rat plasma after infusion.

Receptor-mediated uptake of lysosomal enzymes.

This paper reviews the evidence for two different forms of carbohydrate-mediated recognition of lysosomal enzymes by cell surface receptors. The recognition marker on lysosomal enzymes which are rapidly pinocytosed by fibroblasts contains phosphate, probably linked to D-mannose as a phosphomannosyl moiety. The fraction of lysosomal hydrolases bearing this recognition marker for fibroblasts varies, depending on the tissue source. Another form of recognition accounts for rapid plasma clearance of infused lysosomal enzymes in the rat. This rapid clearance is mediated by receptors on Kupffer cells and other reticuloendothelial cells that recognize mannosyl residues on the glycoprotein hydrolases.

Antigenic structure of hepatitis B surface antigen: identification of the "d" subtype determinant by chemical modification and use of monoclonal antibodies.

Hepatitis B surface antigens (HBsAg) of both the adw and ayw subtypes were reductively methylated with formaldehyde in the presence of sodium cyanoborohydride. The effect on antigenicity was determined by radioimmunoassay with monoclonal antibodies specific for seven different antigenic determinants. The reaction was shown to eliminate specifically the "d" antigenic activity of HBsAg/adw and to have no effect on HBsAg/ayw. Moreover, the reaction had only a slight affect on HBsAg/adw at one of the "a" antigenic determinants. The sites of modification were determined and the extent of modification of each site was compared to the loss of "d" antigenic activity. These studies demonstrated that the loss of "d" activity was due to the modification of lysine 122 in HBsAg/adw, and that although the amino terminus and lysine residues 141 and 160 of both HBsAg/adw and HBsAg/ayw are reactive, their modification does not alter any measurable antigenic activity.

Improved detection of anti-HCV in post-transfusion hepatitis by a third-generation ELISA.

The sensitivity of ORTHO HCV 3.0 ELISA Test System (ELISA 3) for the detection of anti-HCV was compared with the second-generation ELISA, OR-THO HCV 2.0 ELISA Test System (ELISA 2). ELISA 3 differs from ELISA 2 in that it incorporates the HCV recombinant antigen NS5, in addition to recombinant antigens derived from the NS3, NS4 and core regions of the HCV genome. Specimens tested consisted of serial bleeds obtained from 21 individuals undergoing seroconversion following acquisition of post-transfusion HCV infection. ELISA 3 demonstrated significantly greater sensitivity than ELISA 2, detecting seroconversion earlier in 24% (5/21) of cases. Although one of these cases appeared to represent early seroconversion to NS5, most of the improved sensitivity of ELISA 3 appeared to derive from increased detectability of anti-c33c.

Quality control for qualitative assays: quantitative QC procedure designed to assure analytical quality required for an ELISA of hepatitis B surface antigen.

An assay for hepatitis B surface antigen (HBsAg) should reliably detect 0.2 microgram/L, the lowest reported concentration in an asymptomatic blood donor. The difference between this concentration and the assay cutoff defines the analytical quality requirement in a total error format. The design of a statistical QC procedure is critically dependent on the precision of the assay. The precision of a developmental ELISA of HBsAg under study ranged from 17.5% to 9.6% for controls containing 0.07 to 1.50 micrograms/L, respectively. Use of one positive control with the 1(3s), QC rule provided an 85% chance of detecting a critical loss of assay sensitivity; use of two positive controls increased the chance of detecting critical loss of assay sensitivity to nearly 100%. These rules are based on the precision of this developmental assay, and must be developed individually for other assays. The development of the proposed QC procedures illustrates how quantitative QC can be provided for qualitative assays.

Increased detection of hepatitis C virus infection in commercial plasma donors by a third-generation screening assay.

BACKGROUND: Routine screening of blood donations with second-generation hepatitis hepatitis C virus (HCV) assays has substantially reduced the occurrence of posttransfusion hepatitis. However, following the development of third-generation assays, several studies indicated that these assays may identify HCV-infected individuals who are not identified by second-generation assays. STUDY DESIGN AND METHODS: The sensitivity of a third-generation HCV enzyme-linked immunosorbent assay (ELISA-3) was compared with a second-generation ELISA (ELISA-2) in a side-by-side study of 9936 commercial blood donors. ELISA-reactive specimens were subjected to supplemental analysis by third-generation recombinant immunoblot assay and polymerase chain reaction. RESULTS: ELISA-3 demonstrated greater sensitivity than ELISA-2, detecting 1 additional recombinant immunoblot assay-positive specimen per 2000 tested. ELISA-3 also detected 1 additional HCV-infectious polymerase chain reaction-positive unit among approximately 10,000 units screened. CONCLUSION: The incremental sensitivity achieved with ELISA-3 can be expected to eliminate approximately 20 infectious donations per week among those made by commercial donors in the United States. In accordance with previous studies, most of the improved sensitivity of ELISA-3 derives from its increased detection of anti-c33c (NS3), rather than from the inclusion of HCV antigen NS5.