Properties of an Acid Phosphatase in Pulmonary Surfactant (Lung Surfactant/Phosphatidate Phosphatase/Phosphatidylglycerol Phosphate) BRADLEY J

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Proc. Natl. Acad. Sci. USA Vol. 77, No. 2, pp. 808-811, February 1980 Biochemistry Properties of an acid phosphatase in pulmonary surfactant (lung surfactant/phosphatidate phosphatase/phosphatidylglycerol phosphate) BRADLEY J. BENSON Cardiovascular Research Institute, and the Department of Biochemistry, University of California, San Francisco, California 94143 Communicated by John A. Clements, November 5, 1979

ABSTRACT Lung surfactant, a lipid-protein complex pu- phatase (phosphatidate phosphohydrolase, EC 3.1.3.4) in in- rified from dog lungs, contains a highly active phosphomo- tracellular lamellar bodies. Spitzer et al. (13) also found the noesterase associated with it. This phosphatase is quite specific enzyme in their preparations of isolated lamellar bodies, for the hydrolysis of phosphatidic acid and 1-acyl-2-lysophosphatidic acid. The enzyme possesses many of the characteristics whereas Garcia et al. (14) found no phosphatidate phosphatase of the microsomal enzyme, phosphatidate phosphohydrolase in their lamellar body preparations that they could not attribute (EC 3.1.3.4). In addition, we have shown that this enzyme will to microsomal contamination. Recently, however, Spitzer and also convert phosphatidylglycerol phosphate [1(3-sn-phospha- Johnston (15) demonstrated convincingly that the phosphatidate tidyl)sn-glycerol-1-PJ to phosphatidylglycerol [1{3-sn-phos- phosphatase in their lamellar body preparations was not con- phatidyl)sn-glycerolj and Pi. The phosphatidylglycerol phos- tamination from microsomes. Baranska and van Golde (16) phate was made available to the surfactant enzyme in a coupled assay by hydrolysis of cardiolipin [1(3-sn-phosphatidyl)3(3- reported that their preparations of lamellar bodies contained sn-phosphatidyl)sn-glycerolJ by stereospecific cleavage with no phospholipid biosynthetic enzymes that could not be at- phospholipase C (phosphatidylcholine cholinephosphohydro- tributed to microsomal contamination, although their studies lase, EC 3.1.4.3) from Bacillus cereus. This enzyme has been did not measure phosphatidate phosphatase. previously shown to generate the naturally occurring isomer of During further investigations on the function and charac- phosphatidylglycerol phosphate because it has specificity for terization of extracellular SAM proteins, we found an acid the 3(3-sn-phosphatidyl) group of cardiolipin. Other properties of the surfactant enzyme are discussed in relation to its presence phosphomonoesterase that could cleave phosphatidic acid to in lung surface active material. diglyceride and Pi. * This enzyme can also convert the immediate precursor of PtdGro, phosphatidylglycerol phosphate Lung surfactant or surface active material (SAM) is a lipid- (PtdGro-P) to PtdGro and Pi. We report here additional protein complex obtained by broncho-alveolar lavage (1). In properties of this enzyme and suggest that the SAM phosphatase vio this material is thought to adsorb to the air/liquid interface may play a role in phosphatidylglycerol biosynthesis. of the terminal air spaces and, by reducing the surface tension at low lung volumes, contribute to the stability of the alveoli (2, MATERIALS AND METHODS 3). Recent studies from this laboratory have indicated that in The sodium salts of phosphatidate (egg PtdCho), cardiolipin, excised rat lungs this acellular lining layer is capable of main- lysophosphatidate (egg PtdCho), p-nitrophenylphosphate, taining the surface tension in the alveoli below 9 mN/m at glucose-6-phosphate, glycerol-3-phosphate, glycerol-2-phos- functional residual volume (4). phate, p-nitrophenyl-D-mannoside, and p-nitrophenyl-N- Chemical analysis of canine surfactant purified by density acetyl-f3-D-glucosaminide were all obtained from Sigma. Di- gradient centrifugation indicates that it is a complex mixture palmitoylphosphatidic acid, diolein, and dioleoylphosphatidic of lipids and proteins (5). There is general agreement that some acid were supplied by Serdary Biochemicals (London, ON 65% of SAM (wt/wt) is phosphatidylcholine (PtdCho) and, of Canada). Silica gel G and H plates were products of Analtech this, dipalmitoyl PtdCho is the principal molecular species. It (Newark, DE). Chloroform and methanol were redistilled be- is this component of lung surfactant to which the unusual sur- fore use and all water used was double distilled. Other reagents face properties of SAM are attributed (6). The next most were of the highest quality obtainable. abundant class of phospholipid by weight is phosphatidylgly- All lipids were checked for purity by thin-layer chroma- cerol (PtdGro) (7, 8), which is found in uniquely high concen- tography prior to use. (When necessary the lipids were purified tration in SAM, 9-10% by weight. The role of this phospholipid by preparative thin-layer chromatography.) Protein concen- in SAM function is unknown. trations were estimated by the method of Lowry et al. (17), as SAM is thought.to be synthesized by the type II epithelial cells modified by Dulley and Grieve (18). Inorganic phosphorus was of the lungs (9, 10). These cells contain organelles, lamellar measured according to Bartlett (19). bodies, that are thought to be the intracellular sites of surfactant Fresh, excised lungs were obtained from normal, healthy storage prior to secretion into the alveoli. These osmiophilic mixed-breed dogs of either sex. The SAM was prepared essen- inclusion bodies have been purified from different species by tially according to the method of King and Clements (1). The density gradient centrifugation after lung homogenization, and surfactant obtained was centrifuged to equilibrium in a con- their phospholipid composition has been shown to be similar tinuous sodium bromide density gradient (1.057-1.124 g./ml). to that of extracellular SAM (8, 11). The surfactant band was removed and the density was deter- Recently, workers in many laboratories have been studying mined in a pycnometer. After dialysis, aliquots were stored at whether lamellar bodies are only a storage organelle of SAM -700 C. or whether they actively participate in phospholipid biosyn- Force/area isotherm measurements were routinely carried thesis or modification. Meban (12), using electron microscopic out on purified SAM preparations. The lung surfactant was histochemistry, reported the presence of phosphatidate phos- Abbreviations: SAM, surface active material; PtdCho, phosphatidyl- The publication costs of this article were defrayed in part by page choline; PtdGro, phosphatidylglycerol; PtdGro-P, phosphatidylglycerol charge payment. This article must therefore be hereby marked "ad- phosphate. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Benson, B. J. & Clements, J. A. (1976) Am. Chem. Soc. Abstr. Pap. this fact. (Port City Press, Baltimore, MD) 172, Abstr. 165. 808 Downloaded by guest on September 28, 2021 Biochemistry: Benson Proc. Natl. Acad. Sc. USA 77 (1980) 809 dispersed in 66% (vol/vol) isopropanol and spread onto a sub- by chromatography on silica gel G plates developed with pe- phase of 0.9% NaCi solution buffered to pH 7.4 with Tris'HCI. troleum ether/diethyl ether/acetic acid (80:20:1, vol/vol). The The films were confined in a Teflon trough with a tight-fitting, nonpolar lipid released in the reaction migrated with an RF movable Teflon barrier. Surface tension was monitored by a identical to that of synthetic diolein. Negligible (less than 0.5%) platinum dipping plate-straingauge-amplifier system. water-soluble, esterified phosphate and no inorganic phosphorus Phosphatidate phosphatase activity was measured by a were released during the hydrolysis of cardiolipin. modification of the method of Coleman and Hfibscher (20). Other enzymatic activities assayed (substrates) were: acid Dioleoylphosphatidate (0.3 mM) was suspended in 0.16 ml of phosphatase (EC 3.1.3.2) (p-nitrophenylphosphate) (24); reaction mixture of 50 mM sodium maleate (pH 6.5) containing a-mannosidase (EC 3.2.1.24) (p-nitrophenyl-a-D-mannoside) 5-20 jig of protein. The reaction was carried out at 370C for (25); nonspecific esterase (p-nitrophenylacetate) (26); and 30 or 60 min. The reaction was quenched by addition of tri- f3-N-acetylglucosaminidase (EC 3.2.1.53) (p-nitrophenyl-N- chloroacetic acid. After centrifugation at 8000 X g, Pi in the acetyl-f3-D-glucosaminide) (27). Glucose-6-phosphatase was supernatant was measured by a modification of the method assayed according to Coleman and Bell (28). reported by Baginski et al. (21). All assays were linearly proportional to time and protein over the range used in this RESULTS AND DISCUSSION study. Criteria of purity of surfactant PtdGro-P phosphatase activity was measured in a coupled assay system in which PtdGro-P was generated by the action Purified surfactant from canine lungs has a characteristic of phospholipase C according to a modification of the method density of 1.084 g/ml in Tris-buffered saline solutions con- of Cable et al. (22). Cardiolipin (2.5 mg) was incubated with taining sodium bromide to increase the density. Because sur- 1.0 unit of phospholipase C in 0.2 ml of 0.1 M Tris-HCI (pH 7.0) factant is a complex mixture of lipids and proteins, we think it with an aliquot of SAM containing 10,jg of protein. After a 1-hr is important to test each preparation against certain criteria of incubation at 370C, the reaction was stopped by addition of 175 purity before use: the phospholipid-to-protein ratio (wt/wt) Ail of 15% trichloroacetic acid. After centrifugation, the Pi re- should be near 10; the material must possess the antigen pre- leased was then assayed (21). The assay is shown schematically viously shown in this laboratory to be lung- and SAM-specific in Fig. 1. (29); the preparation must be able to form a surface film that, The assay is based on a report by de Haas et al. (23), who on compression, lowers the surface tension of water to below showed that one product of phospholipase C hydrolysis of 9 mN/m (this test is perhaps the most critical because it gives cardiolipin was PtdGro-P and the other product was diacyl- a direct measure of the surfactant "effectiveness" of the ma- glycerol. The phosphorus-containing product was the naturally terial); and the purified SAM preparations also contain about occurring isomer of PtdGro-P because, upon incubation of the 9% by weight PtdGro, a marker that appears to be more specific compound with a cell-free extract from Escherichia coli, than dipalmitoyl PtdCho. PtdGro and Pi were generated. This experiment indicates that We subjected SAM to five additional isopynic bandings and the compound was a substrate for the PtdGro-P phosphatase found that its composition and surface properties were not found in E. coli. Further unequivocal proof for the generation changed by repeated isolation. of PtdGro-P from cardiolipin by phospholipase C treatment is found in a report by Cable et al. (22). They showed that Properties of SAM phosphatase phospholipase C was specific for the 3-(3-sn-phosphatidyl) The pH dependence of the enzyme is shown in Fig. 2. In sodi- group in cardiolipin. By using a stereospecific spin-labeled um maleate buffer, the enzyme was most active at pH 6.5. This analogue of cardiolipin, they were able to show that a product pH optimum is similar to that reported by Coleman and of the phospholipase C hydrolysis had the same stereochemical Hubscher for rat liver microsomes (20), but is slightly different configuration as the naturally occurring PtdGro-P. from that of the phosphatidate phosphatase from lung micro- In other experiments we chromatographed the products of somes (30) and pig lung lamellar bodies (15). the cardiolipin/phospholipase C reaction after lipid extraction The substrate specificity of the SAM phosphatase is shown on silica gel H plates in chloroform/methanol/water/acetic in Table 1. Whereas lysophosphatidate was comparable to acid (65:35:5:14, vol/vol) and detected a very slowly migrating phosphatidate as a substrate for the surfactant enzyme, other phosphorus-containing compound similar to that described by phosphomonoesters were hydrolyzed to a very limited extent Cable et al. (22) and by de Haas et al. (23). The other expected or not at all. The substrate 3-methylumbelliferylphosphate was product of the reaction, diacylglycerol, was shown to be present hydrolyzed to a lesser degree than phosphatidate or lysophos-

HO PLC HO SAM HO phosphatase Cariolp in Ptd + ,D

Cardiolipin PtdGrc PtdGro + diglyceride P. FIG. 1. Schematic representation of the assay for PtdGro-P phosphatase activity. Arrowhead designates the 3 carbon of sn-glycerol; P, phospho; PLC, phospholipase C. Stereochemical representations are from Cable et al. (22). Downloaded by guest on September 28, 2021 810 Biochemistry: Benson Proc. Natl. Acad. Scai. USA 77 (1980) 0.6f crosomal contamination might be present. Arguing against nonspecific microsomal contamination is our observation on the fluid obtained from fetal lamb lungs in utero. We found phosphatidate phosphatase activity in these fluids from lungs that have never been lavaged, so it would appear that secretion of this enzyme occurs in vivo (B. J. Benson and J. A. Clements, c unpublished observations). The specific activity of the phos- 0 0.4 - phatidate phosphatase in our preparations of dog lung microsomes was 18 nmol of Pi per mg per min, about half of the E surfactant source, similar to that reported for pig lung and liver 0 microsomes (15, 31). These data indicate that microsomal contamination is not the only source of the SAM phosphatase. Enzymatic activity as a function of substrate concentration for O. E phosphatidate and lysophosphatidate are shown in Fig. 3. A 0.2 rapid increase in activity for both substrates occurred in the concentration range of 0-0.2 mM. Higher concentrations of both phosphatidate and lysophosphatidate caused a slight de- crease in activity, indicative of enzyme inhibition. The mech- anism of this inhibition is not known. The inhibition by lysophosphatidate could possibly be attributed to the detergent 0 I I I I I properties of this compound at high substrate concentrations. 4. 6 8 Caras and Shapiro (31) and Mavis et al. (30) have also shown pH comparable rates of hydrolysis for phosphatidate and lysophosphatidate in their studies with liver and lung microsomes. FIG. 2. Surfactant phosphatase as a function ofpH. Sodium ac- etate (50 mM) was used to buffer assays between pH 4.0 and 5.4; so- The Km for the surfactant phosphatase is 67 ,uM, and maximal dium maleate (50 mM) was Osed to buffer the assay between pH 6.0 velocity is 35 nmol of Pi per min per mg of protein. No activity and 7.0; and Tris-HCl (50 mM) was used at higher pH. was found when we tested for the presence of lysosomal hy- drolases, a-mannosidase, esterase, and f3-N-acetylglucosaminidase in our SAM preparations. phatidate, but its fluorescent product, 3-methylumbelliferone, The surfactant enzyme displays an apparent requirement makes it a sensitive substrate for assaying phosphatase in very for reduced sulfhydryl groups in that very low concentrations small amounts of lung surfactant such as are found in tracheal of mercuric chloride could inhibit the activity completely fluid of fetal lambs (B. J. Benson and J. A. Clements, unpub- (Table 2). This inhibition could be completely reversed by the lished observations). sulfhydryl-protecting reagent, dithiothreitol. Although the surfactant phosphatase alone did not hydrolyze Because lung surfactant contains unusually large amounts glucose-6-phosphate to any appreciable extent, a small amount of PtdGro, it was of interest to determine whether the surfactant of hydrolysis occurred in the presence of 0.1% Triton X-100. enzyme possessed the capability of generating PtdGro by de- Glucose-6-phosphatase activity is a specific, microsomal marker phosphorylation of its immediate biosynthetic precursor, enzyme (28) that exhibits marked latency. This latency may occur because glucose-6-phosphatase is thought to be located on the luminal face of the microsomal membrane. Therefore, substrate is not accessible to enzyme within the closed microsomal vesicles unless disruptions of the membrane occur, as in the presence of detergent. Such latency was not apparent in the hydrolysis of the other substrates we tested with phosphatidate c phosphatase. These data indicate that a small amount of mi- 4._ 0 cl Table 1. Substrate specificity of SAM phosphatase E 0) 20r Compound Concentration % activity -1 E 0. 1-Acyl-2-lysophosphatidic acid 0.3 mM 102.1 c 0 0 3-Methylumbelliferylphosphate 0.4 mM 28.0 0S p-Nitrophenylphosphate 5 mnM 23.4 10 E *.OA Glucose-6-phosphate 20 mM 3.5 C edw ATP 2-10 mM 1.6 E op-woqq a-Glycerophosphate 5-50 mM ND I f3-Glycerophosphate 5-50 mM ND -20 0 20 40 Phosvitin 2 mg/ml ND 1/mM 3-Methylumbelliferylphosphate and p-nitrophenylphosphate hydrolysis were monitored by changes in fluorescence (excitation, 450 0.1 0.2 0.3 '' 1.0 nm; emission, 350 nm) and absorbance (410 nm), respectively. The concentrations for optimal activity were determined experimentally. Phosphatidate or 1-acyl-2-lysophosphatidate, mM Pi generated in the assay for the other substrates was monitored as FIG. 3. Surfactant phosphatase activity as a function of the described in Material~and Methods. Activities are expressed relative concentration of dioleoylphosphatidate (0) and lysophosphatidate to a specific activity of 31 nmol ofPi per min per mg of protein for the (0). The assay was carried out for 60 min for each concentration of enzyme, with phosphatidate as a substrate. For ATP, a-glycero- substrate. Pi generated was assayed as described in Materials and phosphate, and l3-glycerophosphate, a range of concentrations were Methods. (Inset) Substrate-activity data plotted on a double-re- assayed as indicated. ND, not detectable. ciprocal plot. Downloaded by guest on September 28, 2021 Biochemistry: Benson Proc. Natl. Acad. Sci. USA 77 (1980) 811 Table 2. Effect of mercury and a sulfhydryl-protecting reagent conducting the assays with isolated, purified PtdGro-P under on activity of surfactant phosphatidate phosphatase conditions similar to those with the other substrates. Addition % activity This report indicates that a highly active phosphatase is intimately associated with extracellular SAM. The intracellular SAM phosphatidate phosphatase 100 source of the enzyme remains unclear, but data by Spitzer et +1 mM dithiothreitol 110 al. (15) and Johnston et al. (32) indicate that these enzymes are +1 mM HgC12 0 surfactant-associated intracellularly. Only when phosphatidate +0.5 mM HgCl2 8 phosphatase from various lung-cell organelles has been purified +0.25 mM HgCl2 14 and characterized and contamination by other subcellular +0.25 mM HgC12 fractions quantitated can these questions be answered un- +1 mM dithiothreitol 106 equivocally. HgCl2 was incubated with the surfactant for 5 min prior to initiation ofthe assay. Dithiothreitol was added when the reaction was begun. I thank Dr. John A. Clements for invaluable discussions during this Pi released was measured as described in Materials and Methods. work and Ms. Barbara Ehrlich for preparation of the manuscript. This Activities are relative to a control assay of SAM phosphatidate work was supported by National Institutes of Health Program Project phosphatase for phosphatidate of 32 nmol of Pi per min per mg of Grant HL 06285 and Pulmonary SCOR HL 19185. protein. 1. King, R. J. & Clements, J. A. (1973) Am. J. Physiol. 223, 707- 714. PtdGro-P. As indicated in Fig. 1, the basis for formation of 2. Clements, J. A., Hustead, R. F., Johnson, R. P. & Gribetz, I. (1961) PtdGro-P is the specificity of phospholipase C for the 3-(3- J. Appl. Physiol. 16, 444-450. sn-phosphatidyl) group of cardiolipin. This preference results 3. Pattle, R. E. (1965) Physiol. Rev. 45,48-79. in the formation of the natural stereoisomer of PtdGro-P. The 4. Schurch, S., Goerke, R. J. & Clements, J. A. (1976) Proc. Natl. a substrate Acad. Sci. USA 73, 4698-4702. PtdGro-P generated then simultaneously becomes 5. King, R. J. (1974) Fed. Proc. Fed. Am. Soc. Exp. Biol. 33, for the SAM phosphatase. Results from this experiment are 2238-2247. shown in Table 3. Although PtdGro-P was not hydrolyzed as 6. Goerke, R. J. (1974) Biochim. Biophys. Acta 344,241-261. efficiently as phosphatidate, PtdGro-P was a good substrate for 7. Hallman, M. & Gluck, L. (1975) Biochim. Biophys. Acta 409, the SAM enzyme as well. There are some possible explanations 172-191. for this decreased rate. Although the pH was near optimal for 8. Rooney, S. A., Page-Roberts, B. A. & Motoyama, E. K. (1975) J. the phospholipase C hydrolysis of cardiolipin in the coupled Lipid Res. 16, 418-425. assay, it was not optimal for the SAM phosphatase (pH 6.5). 9. Mason, R. J. (1976) J. Cell Biol. 70, 208. Phospholipase C treatment of the SAM phosphatase gave no 10. Kikkawa, Y., Yoneda, K., Smith, F., Packard, B. & Suzuki, K. release of Pi, indicating that the water-soluble products from (1975) Lab. Invest. 32, 295-302. the SAM (mainly phosphorylcho- 11. Hallman, M. & Gluck, L. (1976) J. Lipid Res. 17, 257-262. hydrolysis of phospholipids 12. Meban, C. (1972) J. Cell Biol. 53, 249-252. line) are not substrates for the SAM phosphatase. The effect of 13. Spitzer, H. L., Rice, J. M., MacDonald, P. C. & Johnston, J. M. removal of some of these groups (usually 20%) on the phos- (1975) Biochem. Biophys. Res. Commun. 66,17-23. phatase cannot be determined quantitatively in this assay with 14. Garcia, A., Sener, S. F. & Mavis, R. D. (1976) Lipids 11, 109- PtdGro-P. In preliminary studies assaying the phosphatase with 112. synthetic substrates, 10-15% of activity was lost by prior con- 15. Spitzer, H. L. & Johnston, J. M. (1978) Biochim. Biophys. Acta version of 50% of the SAM phospholipid to diacylglycerol with 531,275-285. phospholipase C. The substrate concentration of PtdGro-P may 16. Barinska, J. & van Golde, L. M. G. (1977) Biochim. Biophys. Acta not be in the optimal range. For phosphatidate and lysophos- 488,285-293. phatidate, enzymatic rate is directly proportional to substrate 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. concentration. Lower rates of hydrolysis of PtdGro-P would (1951) J. Biol. Chem. 193, 265-275. was low. A less 18. Dulley, J. R. & Grieve, P. A. (1975) Anal. Biochem. 54, 136- be expected if the concentration of this substrate 141. likely possibility is that we are assaying a separate enzyme with 19. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468. this substrate. Arguing against this are the recent data by 20. Coleman, R. & Hubscher, G. (1962) Biochim. Biophys. Acta 56, Johnston e al. (32), who provided convincing evidence that the 479-490. lamellar bodies also possess the ability to convert PtdGro-P to 21. Baginski, E. S., Foa, P. P. & Zak, B. (1967) Clin. Chim. Acta 15, PtdGro. Many of the above questions can be answered by 155-158. 22. Cable, M. B., Jacobus, J. & Powell, G. L. (1978) Proc. Natl. Acad. Sci. USA 75, 1227-1231. Table 3. Hydrolysis of PtdGro-P by SAM phosphatase 23. deHaas, G. H., Bonsen, P. P. M. & Van Deenen, L. L. M. (1966) nmol Pi/min per mg Biochim. Biophys. Acta 116, 114-124. Additions protein 24. Mitchell, R. H., Karnovsky, M. J. & Karnovsky, M. L. (1970) Biochem. J. 116,207-216. Buffer 0 25. Conchie, J. & Hay, A. J. (1959) Biochem. J. 71, 318-325. SAM phosphatase 26. Vatter, A. E., Reiss, 0. K., Newman, J. K., Lindquist, K. & + cardiolipin <0.01 Groeneboer, E. (1968) 1. Cell Biol. 38, 80-98. Phospholipase C (1.0 unit) 27. Sellinger, 0. Z., Beaufay, H., Jacques, P., Doyan, A. & DeDuve, + cardiolipin <0.01 C. (1960) Biochem. J. 74, 450-456. Phospholipase C (1.0 unit) 28. Coleman, R. & Bell, R. M. (1978) J. Cell Biol. 76,245-251. + SAM phosphatase <0.01 29. King, R. J., Gikas, E. G., Ruch, J. & Clements, J. A. (1974) Am. SAM phosphatase Rev. Respir. Dis. 110, 273-281. + cardiolipin 30. Mavis, R. D., Finkelstein, J. N. & Hall, B. P. (1978) J. Lipid Res. + phospholipase C (1.0 19,467-477. 8.1 31. Caras, I. & Shaprio, B. (1975) Biochim. Biophys. Acta 409, unit) 201-211. PtdGro-P was generated in this assay according to de Haas et al. 32. Johnston, J. M., Porter, J. C. & MacDonald, D. C. (1978) in En- (23) as modified by Cable et al. (22). Pi released was measured as zymes in Lipid Metabolism, eds. Gatt, S., Freysz, X. & Mandel, described in Materials and Methods. P. (Plenum, New York), pp. 327-339. Downloaded by guest on September 28, 2021