Guidelines versus evidence: what we can learn from the Australian guideline for low-level laser therapy in knee osteoarthritis? A narrative review.

This narrative review analyses the Australian Guideline (2018) for the treatment of knee osteoarthritis (KOA) developed using Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology. The Guideline recommended against the use low-level laser therapy (LLLT). Why this conclusion was reached is discussed in this review in the context of evidence provided in other systematic reviews, the latest of which was published in 2019 and which provided strong support for LLLT for knee OA. We evaluated the reference list cited for the recommendation "against" LLLT and compared this with reference lists of systematic reviews and studies published before and after the publication date of the Guideline. Eight randomised controlled trials (RCTs) of LLLT were cited in the Guideline the latest of which was published in 2012. There were seventeen additional RCTs, five of which together with one systematic review were located in the year of publication, 2018. The most recent systematic review in 2019 included 22 RCTs in its analysis. Discordance with the levels of evidence and recommendations was identified. Although GRADE methodology is said to be robust for systematically evaluating evidence and developing recommendations, many studies were not identified in the Guideline. In contrast, the latest systematic review and meta-analysis provides robust evidence for supporting the use of LLLT in knee OA. The conflict between guidelines based on opinion and evidence based on meta-analysis is highlighted. Given the totality of the evidence, we recommend that the Australian Guideline should be updated immediately to reflect a "for" recommendation.

Selection and Use of Health Services for Infants' Needs by Indigenous Mothers in Canada: Integrative Literature Review.

In Canada, Indigenous infants experience significant health disparities when compared to non-Indigenous infants, including significantly higher rates of birth complications and infant mortality rates. The use of primary health care is one way to improve health outcomes; however, Indigenous children may use health services less often than non-Indigenous children. To improve health outcomes within this growing population, it is essential to understand how caregivers, defined here as mothers, select and use health services in Canada. This integrative review is the first to critique and synthesize what is known of how Indigenous mothers in Canada experience selecting and using health services to meet the health needs of their infants. Themes identified suggest both Indigenous women and infants face significant challenges; colonialism has had, and continues to have, a detrimental impact on Indigenous mothering; and very little is known about how Indigenous mothers select and use health services to meet the health of their infants. This review revealed significant gaps in the literature and a need for future research. Suggestions are made for how health providers can better support Indigenous mothers and infants in their use of health services, based on what has been explored in the literature to date.

Simple and practical screening approach to identify HIV-infected individuals with depression or at risk of developing depression.

OBJECTIVES: Studies have shown that depression and other mental illnesses are under-diagnosed among HIV-infected individuals. The aim of this study was to evaluate the use of mental health history and questionnaire-based screening instruments to identify HIV-infected individuals at risk of depression. METHODS: The Beck Depression Inventory II (BDI-II) was used to assess the prevalence and severity of depressive symptoms among HIV-infected individuals attending two out-patient clinics in Denmark. HIV-infected individuals with a BDI-II score ≥ 20 were offered a clinical evaluation by a consultant psychiatrist. The BDI-II score was compared to the outcome of mental health history review, and to results obtained using the European AIDS Clinical Society (EACS) two-item depression screening tool. RESULTS: A total of 501 HIV-infected individuals were included in the study. Symptoms of moderate/major depression (BDI-II score ≥ 20) were observed in 111 patients (22%); 65 of these patients consulted a psychiatrist, of whom 71% were diagnosed with a co-existing disorder. The BDI-II score was compared to the outcome of a mental health history review, and to results obtained using the European AIDS Clinical Society (EACS) two-item depression screening tool. The two questions showed a sensitivity and specificity of 95% and 68%, respectively, for diagnosis of current depression or risk of depression. A previous psychiatric history and substance abuse were independently associated with an increased risk of depression. CONCLUSIONS: We suggest that the mental health of HIV-infected individuals should be reviewed and a "risk-flag" three-step approach should be used (1) to screen routinely with the two verbal questions suggested by the EACS, (2) to identify whether there is a risk of depression and then screen with the BDI-II, and (3) to identify whether there is still a risk and then perform a full evaluation and obtain an accurate psychiatric diagnosis by a psychiatrist.

Factors associated with risk of depression and relevant predictors of screening for depression in clinical practice: a cross-sectional study among HIV-infected individuals in Denmark.

OBJECTIVES: Depression and psychiatric disorders are frequent among HIV-infected individuals. The aim of this study was to determine the prevalence of depression and describe the psychiatric history of HIV-infected individuals in an out-patient clinic in Denmark and to identify factors of clinical importance that may be used to identify patients at risk of depression. METHODS: In 2013, 212 HIV-infected patients were included in a questionnaire study. We used the Beck Depression Inventory II (BDI-II) to assess the prevalence and severity of depressive symptoms. Patients with a BDI-II score ≥ 20 were offered a clinical evaluation by a consultant psychiatrist. Logistic regression was used to determine predictors associated with risk of depression. RESULTS: Symptoms of depression (BDI-II score ≥ 14) were observed in 75 patients (35%), and symptoms of moderate to major depression (BDI-II score ≥ 20) in 55 patients (26%). There was also a high prevalence of co-occurring mental illness. In a multivariate model, self-reported stress, self-reported perception that HIV infection affects all aspects of life, self-reported poor health, not being satisfied with one's current life situation, previous alcohol abuse, nonadherence to antiretroviral therapy and previously having sought help because of psychological problems were independently associated with risk of depression. CONCLUSIONS: Symptoms of depression and co-occurring mental illness are under-diagnosed and under-treated among HIV-infected individuals. We recommend that screening of depression should be conducted regularly to provide a full psychiatric profile to decrease the risk of depression and improve adherence and quality of life in this population.

Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells.

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38alpha. Mice null for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38alpha(1/+) ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38alpha, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38alpha(-/)- ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38alpha(1/+) but not p38alpha(-/)- IL-1R-positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells. Therefore, these studies demonstrate that p38alpha is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38alpha does not serve an indispensable role in apoptosis.

Ytterbium-doped fibre femtosecond laser offers robust operation with deep and precise microsurgery of C. elegans neurons.

Laser microsurgery is a powerful tool for neurobiology, used to ablate cells and sever neurites in-vivo. We compare a relatively new laser source to two well-established designs. Rare-earth-doped mode-locked fibre lasers that produce high power pulses recently gained popularity for industrial uses. Such systems are manufactured to high standards of robustness and low maintenance requirements typical of solid-state lasers. We demonstrate that an Ytterbium-doped fibre femtosecond laser is comparable in precision to a Ti:Sapphire femtosecond laser (1-2 micrometres), but with added operational reliability. Due to the lower pulse energy required to ablate, it is more precise than a solid-state nanosecond laser. Due to reduced scattering of near infrared light, it can lesion deeper (more than 100 micrometres) in tissue. These advantages are not specific to the model system ablated for our demonstration, namely neurites in the nematode C. elegans, but are applicable to other systems and transparent tissue where a precise micron-resolution dissection is required.

Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor.

Mouse L-cells that contain the cation-independent (CI) mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor endocytose acid hydrolases and deliver these enzymes to lysosomes. The postendocytic loss of the Man 6-P recognition marker from the cell-associated acid hydrolases was assessed by CI-Man 6-P receptor affinity chromatography. 125I-labeled acid hydrolases internalized by L-cells grown at high density were delivered to lysosomes but were not dephosphorylated. In contrast, the same 125I-labeled hydrolases internalized by L-cells maintained at low density were delivered to lysosomes and were extensively dephosphorylated. The dephosphorylation at low density required 5 h for completion suggesting that the phosphatase responsible for the dephosphorylation is located within the lysosomal compartment. Transition from the high to low density state was rapid and was not inhibited by cycloheximide. Medium substitution experiments indicated that serum factors were necessary to maintain the L-cells in the dephosphorylation-competent (low density) state, and that serum-free conditions led to a dephosphorylation-incompetent (high density) state. Addition of IGF II to cells in serum-free medium allowed acid hydrolases subsequently introduced by endocytosis to be dephosphorylated. The results indicate that the removal of the Man 6-P recognition marker from endocytosed acid hydrolases is regulated by serum factors in the growth medium, including IGF II.

Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization.

Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum (Einstein, R., and C. A. Gabel. 1989. J. Cell Biol. 109:1037-1046). To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated. This difference in processing indicates that lysosomes themselves exist in a dephosphorylation-competent and -incompetent state. Man 6-P-bearing acid hydrolases endocytosed by the L+ cells in the absence of serum were not distributed uniformly throughout the lysosomal compartment. The change in the dephosphorylation competence of L cells in response to serum suggests, therefore, that these cells contain multiple populations of lysosomes that differ with respect to their content of a mannose 6-phosphatase, and that serum factors affect the distribution of hydrolases between the different compartments.

Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells.

The murine plasma cell line MOPC 315 efficiently targets newly synthesized acid hydrolases to lysosomes in spite of a marked deficiency in the level of the mannose 6-phosphate receptor (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). To better understand the routing of lysosomal enzymes in this cell line, pulse-chase experiments were performed with [2-3H]mannose and [35S]methionine followed by immunoprecipitation of beta-glucuronidase and IgA. By 3 h of chase, essentially all of the newly synthesized beta-glucuronidase had undergone proteolytic processing, suggesting that the molecules had reached lysosomes. At this time 30% of the pulse-labeled IgA was still intracellular. The oligosaccharides on the intracellular IgA were of the high mannose-type, while the secreted IgA contained processed, complex-type oligosaccharides. This indicates that the intracellular IgA was still in the endoplasmic reticulum or an early region of the Golgi complex when all of the beta-glucuronidase had reached lysosomes. Therefore, beta-glucuronidase and IgA must exit from the endoplasmic reticulum or the early Golgi complex at different rates, a finding that is inconsistent with bulk phase movement of these proteins from the endoplasmic reticulum to the trans Golgi complex. The addition of the ionophore monensin greatly slows the rate of IgA secretion from MOPC 315 cells and the molecules secreted have incompletely processed oligosaccharides. In contrast, monensin only slightly delays the transport of newly synthesized beta-glucuronidase to lysosomes and causes no significant alteration in the extent of oligosaccharide phosphorylation, a process that appears to occur in the early (cis) Golgi complex. However, the labeled beta-glucuronidase was deficient in sialylated, phosphorylated hybrid oligosaccharides whose biosynthesis requires the action of late stage oligosaccharide processing enzymes assumed to be localized in the trans Golgi complex.

Mannose 6-phosphate receptor-mediated endocytosis of acid hydrolases: internalization of beta-glucuronidase is accompanied by a limited dephosphorylation.

Endocytosis of acid hydrolases via the cell surface mannose 6-phosphate (Man 6-P) receptor results in the delivery of the enzymes to lysosomes. To examine the fate of the ligand-associated phosphorylated high mannose oligosaccharides, we have analyzed the asparagine-linked oligosaccharides attached to beta-glucuronidase after uptake and processing by Man 6-P receptor-positive mouse L cells. beta-Glucuronidase, double-labeled with [2-3H]mannose and [35S]methionine, was isolated from the growth medium of mouse P388D1 cells. 80% of the [3H]mannose associated with the secreted enzyme was recovered as high mannose-type oligosaccharides, and 24-37% of these units were phosphorylated. Three species of phosphorylated oligosaccharides were identified; high mannose-type units containing either one or two phosphomonoesters, and hybrid-type units containing one phosphomonoester and one sialic acid residue. After endocytosis by the L cells, the beta-glucuronidase molecules migrated faster on an SDS gel, suggesting that the enzymes had been processed within lysosomes. Examination of the cell-associated beta-glucuronidase molecules indicated that: (a) the percentage of phosphorylated oligosaccharides remained comparable to the input form of the enzyme, even after a 24-h chase period, (b) the presence of a single species of phosphorylated oligosaccharide that contained one phosphomonoester, and (c) the positioning of the phosphate within the intracellular monophosphorylated species was comparable to the positioning of the phosphate within the two phosphomonoester species originally secreted by the P388D1 cells. Therefore, the internalized beta-glucuronidase molecules undergo a limited dephosphorylation; oligosaccharides containing two phosphomonoesters are converted to monophosphorylated species, but the one phosphomonoester forms are conserved. A comparison of the phosphorylated oligosaccharides recovered from ligands internalized by the L cells at 37 degrees and 20 degrees C indicated that: (a) molecules internalized at 20 degrees C retain a higher percentage of phosphorylated structures; and (b) at both temperatures the predominant phosphorylated oligosaccharide contains a single phosphomonoester group. The results indicate that the Man 6-P recognition marker persists after endocytosis and delivery to lysosomes and support the possibility that the limited dephosphorylation of the oligosaccharides may occur en route to these organelles.

Lysosomal enzyme trafficking in mannose 6-phosphate receptor-positive mouse L-cells: demonstration of a steady state accumulation of phosphorylated acid hydrolases.

During their transport from the endoplasmic reticulum to lysosomes, newly synthesized acid hydrolases are phosphorylated in the Golgi apparatus to generate a common recognition marker, mannose 6-phosphate (Man 6-P). The phosphorylated acid hydrolases then bind to the Man 6-P receptor and are transported by an unknown route to lysosomes. To learn more about the delivery pathway, we examined the fate of the phosphorylated oligosaccharides synthesized by a Man 6-P receptor-positive line of mouse L-cells. In contrast to the rapid degradation of the recognition marker previously observed in mouse lymphoma cells (Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536-542), the number of high mannose oligosaccharides phosphorylated by the L-cells after a 30-min pulse labeling with [2-3H]mannose increased continuously during a subsequent 4-h chase period to a maximum of 9.3% of the total cell-associated structures. After 19 h of chase the absolute number of phosphorylated oligosaccharides declined, but the loss was accompanied by a general loss of cellular oligosaccharides such that 7.4% of the cell-associated high mannose oligosaccharides remained phosphorylated. The longevity of the Man 6-P recognition marker in the L-cells was verified by analyzing the ability of an individual acid hydrolase, beta-glucuronidase, to serve as a ligand for the Man 6-P receptor. At least 60% of the steady state beta-glucuronidase molecules isolated from the L-cells could undergo receptor-mediated endocytosis into enzyme-deficient human fibroblasts. Dense lysosomal granules isolated by metrizamide gradient centrifugation from [3H]mannose-labeled L-cells were found to be highly enriched in their content of phosphomonoester-containing oligosaccharides. The data indicate that acid hydrolases may retain their Man 6-P recognition markers within lysosomes, and suggest the possibility that dephosphorylation occurs at a nonlysosomal location through which the newly synthesized enzymes pass en route to lysosomes.

Processing of the asparagine-linked oligosaccharides of secreted and intracellular forms of the vesicular stomatitis virus G protein: in vivo evidence of Golgi apparatus compartmentalization.

The structures of the asparagine-linked oligosaccharides of several variant forms of the vesicular stomatitis virus glycoprotein transiently expressed from cloned cDNAs have been determined. Glycopeptides isolated from forms of the G protein that reach the cell surface or that are secreted into the medium are virtually identical; they contain complex-type oligosaccharides whose nonreducing ends terminate in galactose and sialic acid residues. In contrast, forms of the G protein that remain intracellular possess oligosaccharides at intermediate stages in the processing pathway. One deletion mutant, delta 1473, codes for a protein that remains in the rough endoplasmic reticulum (Rose, J. K., and J. E. Bergmann, 1982, Cell, 30:753-762) and contains only high mannose-type oligosaccharides. Another mutant, delta 1554, codes for a glycoprotein that contains oligosaccharides of primarily two classes. One class is of the high mannose type and is similar to those found on the protein coded for by delta 1473. However, the major class contains biantennary and more highly branched complex-type oligosaccharides that terminate in N-acetylglucosamine rather than galactose or sialic acid residues. These data suggest that the protein coded for by delta 1554 migrates to the Golgi apparatus, but does not enter the more distal compartment(s) of the organelle which contains galactosyl- and sialyltransferases.

Postendocytic maturation of acid hydrolases: evidence of prelysosomal processing.

The mannose 6-phosphate (Man 6-P) receptor operates to transport both endogenous newly synthesized acid hydrolases and extracellular enzymes to the lysosomal compartment. In a previous study (Gabel, C. A., and S. A. Foster, 1986, J. Cell Biol., 103:1817-1827), we noted that beta-glucuronidase molecules internalized by mouse L-cells via the Man 6-P receptor undergo a proteolytic cleavage and a limited dephosphorylation. In this report, we present evidence that indicates that the postendocytic alterations of the acid hydrolase molecules occur at a site through which the enzymes pass en route to the lysosomal compartment. Mouse L-cells incubated at 20 degrees C with beta-glucuronidase (isolated from mouse macrophage secretions) internalize the enzyme in a process that is inhibited by Man 6-P but unaffected by cycloheximide. As such, the linear accumulation of the ligand observed at 20 degrees C appears to occur through the continued recycling of the cell surface Man 6-P receptor. The subcellular distribution of the internalized ligands was assessed after homogenization of the cells and fractionation of the extracts by density gradient centrifugation. In contrast to the accumulation of the ligand within lysosomes at 37 degrees C, the beta-glucuronidase molecules internalized by the L cells at 20 degrees C accumulate within a population of vesicles that sediment at the same density as endocytic vesicles. Biochemical analysis of the internalized ligands indicates that: (a) the subunit molecular mass of both beta-glucuronidase and beta-galactosidase decrease upon cell association relative to the input form of the enzymes, and (b) the beta-glucuronidase molecules experience a limited dephosphorylation such that high-mannose-type oligosaccharides containing two phosphomonoesters are converted to single phosphomonoester forms. The same two post-endocytic alterations occur after the internalization of beta-glucuronidase by human I-cell disease fibroblasts, despite the low acid hydrolase content of these cells. The results indicate, therefore, that acid hydrolases internalized via the Man 6-P receptor are processed within the endocytic compartment. In that endogenous newly synthesized acid hydrolases display similar alterations during their maturation, the results further suggest that the endosomal compartment is involved in the sorting of ligands transported via both the cell surface and intracellular Man 6-P receptor.

Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.

Lysosomal enzyme oligosaccharide phosphorylation in mouse lymphoma cells: specificity and kinetics of binding to the mannose 6-phosphate receptor in vivo.

Phosphomannosyl residues on lysosomal enzymes serve as an essential component of the recognition marker necessary for binding to the mannose 6-phosphate (Man 6-P) receptor and translocation to lysosomes. The high mannose-type oligosaccharide units of lysosomal enzymes are phosphorylated by the following mechanism: N-acetylglucosamine 1-phosphate is transferred to the 6 position of a mannose residue to form a phosphodiester; then N- acetylglucosamine is removed to expose a phosphomonoester. We examined the kinetics of this phosphorylation pathway in the murine lymphoma BW5147.3 cell line to determine the state of oligosaccharide phosphorylation at the time the newly synthesized lysosomal enzymes bind to the receptor. Cells were labeled with [2-(3)H]mannose for 20 min and then chased for various times up to 4 h. The binding of newly synthesized glycoproteins to the Man 6-P receptor was followed by eluting the bound ligand with Man 6-P. Receptor-bound material was first detected at 30 min of chase and reached a maximum at 60 min of chase, at which time approximately 10 percent of the total phosphorylated oligosaccharides were associated with the receptor. During longer chase times, the total quantity of cellular phosphorylated oligosaccharides decreased with a half-time of 1.4 h, suggesting that the lysosomal enzymes had reached their destination and had been dephosphorylated. The structures of the phosphorylated aligosaccharides of the eluted ligand were then determined and compared with the phosphorylated oligosaccharides of molecules which were not bond to the receptor. The major phosphorylated oligosaccharide species present in the nonreceptor-bound material contained a single phosphosphodiester at all time examined. In contrast, receptor-bound oligosaccharides were greatly enriched in species possessing one and two phosphomonoesters. These results indicate that binding of newly synthesized lysosomal enzymes to the Man 6-P receptor occurs only after removal of the covering N- acetylglucosamine residues.

Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines.

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.