(Received for publication, December 19, 1994)
From the
The lymphocyte adhesion molecule CD44 recognizes a non-hyaluronate proteoglycan, gp600, secreted by mouse T cell line CTLL2. We now demonstrate that gp600 is identical to serglycin, a member of the small proteoglycan family stored in intracellular secretory granules of lymphoid, myeloid, and some tumor cells. Purified gp600 has the ability to bind specifically to CD44, and the binding is dependent on activation of CD44. The CD44-binding elements on gp600 or serglycin are glycosaminoglycans consisting of chondroitin 4-sulfate. Serglycin is readily exocytosed, and its interaction with active form CD44 augments the CD3-dependent degranulation of CD44 positive CTL clones. We conclude that the serglycin secreted from secretory granules of hematopoietic cells is a novel ligand for CD44, and could regulate lymphoid cell adherence and activation.
CD44, which exhibits significant sequence homology to the phylogenically conserved amino-terminal domain of cartilage link proteins, is an important cell surface adhesion molecule expressed on lymphoid cells, myeloid cells, fibroblasts, epithelial cells, and endothelial cells (Jalkanen et al., 1986; Stamenkovic et al., 1989; Goldstein et al., 1989; for reviews, see Haynes et al.(1989) and Lesley et al. (1993a)). Recent studies reveal that this molecule has many isoforms with various inserts in the membrane proximal portion (for review, see Herrlich et al.(1993)). Ligands for CD44 have been shown to be extracellular matrix components such as hyaluronic acid (Aruffo et al., 1990), fibronectin (Jalkanen and Jalkanen, 1992), and collagen types I and VI (Wayner et al., 1987; Carter and Wayner, 1988). In addition, the chondroitin sulfate-modified invariant chain has recently been suggested to be a ligand for CD44 (Naujokas et al., 1993). The CD44 molecule is thought to participate in various adhesive events including lymphocyte recirculation (Jalkanen et al., 1987), lymphohemopoiesis (Miyake et al., 1990), and tumor cell invasiveness (Gunthert et al., 1991). Monoclonal antibodies directed against the CD44 molecule enhance the proliferation of T cells (Shimizu et al., 1989; Denning et al., 1990). In addition, some anti-CD44 monoclonal antibodies can trigger effector functions of murine and human T cell clones (Seth et al., 1991; Galandrini et al., 1993). Therefore, one of the important functions of CD44 in the immune system seems to be the activation of lymphocytes, although the natural ligand interacting with CD44 remains to be determined.
An anti-CD44 monoclonal antibody, Hermes-3, that does not interfere with hyaluronate binding (Culty et al., 1990), inhibits the binding of human lymphocytes to high endothelial venules on frozen lymph node sections, indicating the involvement of CD44 in lymphocyte homing (Jalkanen et al., 1987). It was also reported that the binding of murine lymphocytes to high endothelial venules is resistant to hyaluronidase treatment (Culty et al., 1990), indicating that hyaluronate is not a ligand for CD44 in lymphocyte-high endothelial venule interaction. In the search for a novel ligand for CD44, we identified a sulfated macromolecule, gp600, in the culture supernatant of a murine T cell line, and reported that gp600 is a proteoglycan consisting of a small core protein (18-22 kDa) and chondroitin sulfate-like glycosaminoglycans (Toyama-Sorimachi and Miyasaka, 1994).
Proteoglycans found ubiquitously in tissues are composed of a
protein core and glycosaminoglycan side chains (for reviews, see
Ruoslahti (1989) and Kolset and Gallagher(1990)). In hematopoietic
cells, a member of the proteoglycan family termed serglycin is found in
the secretory granules (for review, see Stevens et al.(1988)).
Serglycins can be classified into 2 groups on the basis of the nature
of the glycosaminoglycans, heparan sulfate serglycins and chondroitin
sulfate serglycins. Various types of serglycin have been characterized, e.g. a heparin sulfate type in serosal mast cells, a
chondroitin 4,6-sulfate type in mouse bone marrow-derived mast cells, a
chondroitin 4-sulfate type in natural killer cells, eosinophils, and
HL-60 leukemic promyelocytes, and a chondroitin 6-sulfate type in
megakaryocytes and platelets (Stevens et al., 1988a; Kolset
and Gallagher, 1990). Serglycin is distinct from all other cell
surface- and matrix-localized proteoglycans both in its high degree of
sulfation and its resistance to proteolysis. Although the molecular
masses of these proteoglycans are heterogenous (60-750 kDa) due
to differences in their glycosaminoglycan side chains, the gene
responsible for the peptide core, which is composed primarily of tandem
serine-glycine repeats, is a single gene (Tantravahi et al.,
1986). The serglycin peptide core is estimated to be M 16,000-18,000 (Bourdon et al., 1985; Stevens et al., 1988b; Avraham et al., 1989), similar to that
of gp600 (Toyama-Sorimachi and Miyasaka, 1994). The expression of
serglycin seems to be restricted to the yolk sac, hematopoietic cells,
and some tumor cells. Serglycin has been suggested to participate in
the packaging of basically charged serine proteases in secretory
granules and the regulation of their enzymatic activity (Stevens et
al., 1988a). It has also been postulated that serglycin plays a
role in cell-mediated cytotoxicity, since these proteoglycans are
exocytosed when an effector cell kills tumor target cells (MacDermott et al., 1985). However, the functions of serglycin have not
been fully defined.
In the present study, since the various characteristics of gp600 so far identified remarkably resemble those of chondroitin sulfate serglycin, we isolated and biochemically characterized gp600 in detail to determine whether gp600 is indeed serglycin. We show that the amino acid sequence of the core protein of purified gp600 is identical to that of serglycin and that chondroitin 4-sulfate, a major glycosaminoglycan of gp600, is essential for CD44 binding. Furthermore, we indicate that CD44-serglycin interaction is involved in lymphoid cells adhesiveness and activation. This study provides further understanding not only of the physiological functions of CD44 but also those of serglycin.
CTLL2, CTLL2 transfectants
(Toyama-Sorimachi and Miyasaka, 1994) of mouse CD44, and mouse thymoma
cell line BW5147 were grown in RPMI 1640 supplemented with 10% fetal
calf serum (Iansa), 10 mM Hepes, 2 mML-glutamine, 1 mM sodium pyruvate,
10M 2-mercaptoethanol, 1% (v/v) 100
nonessential amino acids (Flow Laboratories), 100 units/ml
penicillin, and 100 µg/ml streptomycin (complete medium). For the
culture of CTLL2 and its transfectants, 1 nM recombinant mouse
interleukin-2 was added to the complete medium (Karasuyama et
al., 1989). In the case of large scale culture of CTLL2,
serum-free medium EX-cell 300
(JRH Bioscience) was used.
The culture of bone marrow-derived mast cells was performed as
described previously (Razin et al., 1984).
Figure 1:
DEAE-ion exchange chromatography
profile of gp600 obtained by hydroxylapatite chromatography. The gp600
fraction from hydroxylapatite chromatography was applied to a TSKG-DEAE
column. After washing, the column was eluted at 20 °C with a linear
gradient of NaCl as indicated. Fractions were analyzed for uronic acid; A (
), A
(
),
and A
(
).
Figure 2:
Binding of soluble CD44 to purified gp600.
Increasing amounts of purified gp600 were immobilized on an ELISA
plate, and the binding of CD44-IgG () and control human IgG
(
) to gp600 was assessed as described under ``Experimental
Procedures.''
We then examined CD44 binding to
gp600 by a cell binding assay. CD44 positive or negative cell lines
were labeled with fluorescent dye, and the adherence of cells to
immobilized gp600 was assessed by measuring fluorescence intensity.
CD44 positive BW5147 cells adhered to both gp600 and hyaluronate in the
absence of Ca and Mg
cations (Fig. 3). In contrast, CD44 negative CTLL2 cells failed to
adhere to either gp600 or hyaluronate, but transfection of CTLL2 cells
with CD44 cDNA (Toyama-Sorimachi and Miyasaka, 1994) resulted in a
marked increase in adhesion. The binding of CD44 positive cells to both
gp600 and hyaluronate was completely inhibited by an anti-CD44
monoclonal antibody KM201 (Fig. 3) but not by control rat IgG or
isotype-matched monoclonal antibodies (data not shown), indicating that
cell adhesion to gp600 is mediated by CD44. Hyaluronidase had no effect
on the binding of CD44 positive cell lines to gp600, although the
binding to hyaluronate was completely eliminated by enzyme treatment (Fig. 3, right column), clearly indicating that
hyaluronate is not involved in the interaction between CD44 and gp600.
Figure 3: CD44-dependent cell adhesion to immobilized gp600. CD44 positive (BW5147 and CTLL2MCD44-1) and negative (CTLL2) cell lines were labeled with fluorescent dye, and their binding to immobilized gp600 (left column) or hyaluronate (right column) was examined. Lane 1, control binding to immobilized materials in the absence of anti-CD44; lane 2, lane 1 plus 50 µg/ml anti-CD44 (KM201); lane 3, hyaluronidase treatment of immobilized materials.
Figure 4:
Identification of gp600 core protein as
serglycin. A, preparation of the gp600 core protein by
degradation of the glycosaminoglycans with chondroitinase ABC. The core
protein of gp600 was radiolabeled with NaI by the
lactoperoxidase method. A small amount of radiolabeled gp600 (10
cpm) was mixed with 1 mg of purified, unlabeled gp600 for
detection of the core protein. After overnight treatment with
chondroitinase ABC, the core protein was precipitated by adding
acetone/ethanol. Gp600 (
) or gp600 treated with chondroitinase
ABC (
) was applied to a gel filtration column. Fractions (No.
39-46) from gel filtration chromatography were pooled for
NH
-terminal amino acid sequence analysis. B,
comparison of the amino acid sequences (single letter code) of the
gp600 core protein with serglycin peptide cores expressed in mouse bone
marrow-derived mast cells (BMMC), rat basophilic leukemia-1 (RBL) cells, and human HL-60 cells. Numbers indicate
the residue numbers in the respective
sequences.
Serglycin is known to localize in the secretory granules of granular leukocytes. Therefore, we examined immunohistologically whether soluble CD44 binds to intracellular secretory granules in CTLL2. It was revealed that CD44-IgG stained the cytoplasmic granules of CTLL2 distinctly while no significant fluorescence was observed with control human IgG (Fig. 5). A similar observation was obtained with interleukin-3-dependent mouse bone marrow-derived mast cells (Fig. 5), which produce chondroitin sulfate-type serglycin in their secretory granules (Razin et al., 1982; Stevens et al., 1985). These results support the notion that CD44 binds to serglycin.
Figure 5: Immunofluorescence staining of intracellular granules of CTLL2 and mouse bone marrowderived mast cells with CD44-IgG. Cytospin samples of cells fixed with 3% formalin were incubated with 10 µg/ml CD44-IgG or control human IgG.
Figure 6:
Quantitation of CD44-IgG binding to gp600
by ELISA after treatment with mucopolysaccharide degrading enzymes. A, binding of CD44-IgG to immobilized gp600. Purified gp600
(100 µg/ml) was coated on plastic plates before (lane 1)
or after treatment with chondroitinase ABC (lane 2),
chondroitinase ACII (lane 3), chondroitinase ABC in the
presence of 100 mM Zn and 1 mg/ml dermatan
sulfate (lane 4), hyaluronidase (lane 5), heparinase (lane 6), heparitinase (lane 7), or fucosidase (lane 8). In lane 9, BSA was used instead of gp600.
CD44-IgG binding was examined as described under ``Experimental
Procedures.'' B, binding of CD44-IgG to various
glycosaminoglycans. ELISA plates were coated with 1 mg/ml purified
gp600 (lane 1), chondroitin (lane 2), hyaluronate (lane 3), chondroitin 4-sulfate (lane 4), chondroitin
6-sulfate (lane 5), dermatan sulfate (lane 6),
heparan sulfate (lane 7), keratan sulfate (lane 8),
keratan polysulfate (lane 9), heparin (lane 10), or
BSA (lane 11) at 4 °C
overnight.
To investigate the kind of chondroitin sulfate involved in CD44 recognition, disaccharide analysis was performed. Gp600 was completely digested with chondroitinase ABC and the disaccharides obtained were subjected to HPLC analysis. The digest eluted at the position of 4-sulfated disaccharides, and neither non-sulfated nor disulfated disaccharides were detected (Fig. 7). The mass spectrum of the digest also supported this observation (data not shown). These results indicate that gp600 is a chondroitin 4-sulfate type serglycin, and that chondroitin 4-sulfate chains on gp600 are essential for CD44 binding.
Figure 7:
Disaccharide analysis of gp600
glycosaminoglycans by HPLC. The oligosaccharide fraction prepared by
chondroitinase ABC treatment of gp600 was chromatographed on an amino
silica gel column. The elution positions of authentic unsaturated
chondro-disaccharides are indicated. A, oligosaccharide
obtained from gp600; B, A plus authentic Di-4S.
Disaccharide standard used are:
Di-0S,
4,5-GlcA(
1-3)GalNAc;
Di-6S,
4,5-GlcA(
1-3)GalNAc(6-O-sulfate);
Di-4S,
4,5-GlcA(
1-3)GalNAc(4-O-sulfate);
Di-diSD,
4,5GlcA(2-O-sulfate)(
1-3)GalNAc(6-O-sulfate);
Di-diSE,
4,5-GlcA(
1-3)GalNAc(4,6-O-sulfate);
Di-triS,
4,4-GlcA(2-O-sulfate)(
1-3)
GalNAc(4,6-O-disulfate).
We next tested the reactivity of CD44-IgG with various chondroitin sulfates by ELISA. Although the binding of CD44-IgG to gp600/serglycin and hyaluronic acid was readily detected, no significant binding to either chondroitin 4-sulfate or chondroitin 6-sulfate was observed (Fig. 6B). Similarly, these chondroitin sulfate preparations did not interfere with the binding of fluoresceinated hyaluronate to CD44-positive BW5147 cells, although gp600 strongly inhibited hyaluronate binding to CD44-positive cells as assessed by flow cytometry. Dose-dependent blockage of hyaluronate binding was observed with gp600, and almost complete blockage was obtained at 500 µg/ml (Table 2). These results suggest that the gp600 binding domain on CD44 overlaps with or is close to the hyaluronate binding portion. Chondroitin sulfates A and E were slightly effective at 500 µg/ml, but other chondroitin sulfates were inactive. The finding that none of the defined chondroitin 4-sulfates tested so far was recognized by CD44 suggests that the association of chondroitin sulfates with the core protein is important for CD44 binding.
Figure 8:
CD44-gp600 interaction is involved in
lymphoid cell adherence and activation. Cells were pretreated with
IRAWB14 antibody at a concentration of 10 µg/ml to induce ligand
binding activity of CD44. A, CD44-dependent binding of
peripheral lymphocytes to gp600. This was examined in the presence or
absence of blocking anti-CD44 antibody, KM201(20 µg/ml), or
hyaluronate (1 mg/ml). B, granzyme A release of CTL clone.
Anti-CD3 monoclonal antibody (2C11) was immobilized on a plastic plate
at a concentration of 0.2 µg/ml, and the granzyme A release assay
was performed using mouse CTL clone 5-57 in the presence of gp600
() or hyaluronate (
). Augmentation of granzyme release was
not observed in the absence of gp600 (
) or IRAWB14
(
).
Second, the effect of gp600 on cytotoxic T cell (CTL) activation was examined, since the ligation of cell-surface CD44 has been shown to lead to T cell proliferation (Shimizu et al., 1989; Denning et al., 1990) and activation of CTL (Seth et al., 1991; Galandrini et al., 1993). Inasmuch as the CD44 expressed on CTL clones that we used was also inactive and did not bind to hyaluronate or gp600, we pretreated the clones with IRAWB14. After treatment, CD44 expressed on CTL clones could recognize hyaluronate and gp600 (data not shown). Under these conditions, anti-CD3 induced granzyme A release was examined in the presence or absence of gp600. As shown in Fig. 8, the addition of gp600 to CTL clones significantly enhanced anti-CD3 induced granzyme A release. Treatment with IRAWB14 alone had no effect on the CD3-dependent granzyme release (Fig. 8B, closed square). The enhancement of granzyme release was noticeable especially at suboptimal concentrations of anti-CD3 antibody, and was not observed in the absence of anti-CD3 (data not shown). In contrast to gp600/serglycin, hyaluronate did not enhance CD3-dependent granzyme release at any concentration examined (Fig. 8B). These results suggest that gp600/serglycin can activate CTL in a CD3-dependent manner, and could be important ligand for CD44.
In this report we show that a novel ligand for CD44, gp600,
is a chondroitin 4-sulfate type serglycin stored in secretory granules
of lymphoid and myeloid cells. Based on the following observations, we
conclude that gp600, a novel ligand for CD44, is identical to
serglycin. First, the NH-terminal amino acid sequence of
the gp600 core protein coincides with that of the mouse serglycin core
protein. Second, soluble CD44 recognizes intracytoplasmic secretory
granules where serglycin is known to be present. Third, the molecular
mass of gp600 treated with chondroitinase ABC or ACII is similar to
that of serglycin (10-30 kDa) on SDS-PAGE (Stevens et
al., 1985; Toyama-Sorimachi and Miyasaka, 1994). Fourth,
transcription of the serglycin gene was confirmed in CTLL2 cells by
polymerase chain reaction analysis. In addition, polyclonal antibody
raised against a synthetic serglycin core peptide recognized the gp600
core protein. (
)A specific interaction between CD44 and
gp600 was verified by the following observations: 1) purified gp600
binds dose dependently to CD44-IgG but not to control human-IgG; 2)
purified gp600 binds to CD44-positive cells but not to CD44-negative
cells; 3) the binding is completely inhibited by anti-CD44 monoclonal
antibody; 4) purified gp600 interferes with CD44 binding to
hyaluronate.
The NH-terminal sequence of the gp600 core
protein we evaluated corresponds to the sequence starting from the 84th
residue of the predicted amino acid sequence of mouse serglycin
(Avraham et al., 1989). Similarly, the NH
-terminal
sequence of the rat yolk sac tumor serglycin corresponded to the
sequence from the 75th residue of the predicted amino acid sequence of
rat serglycin (Bourdon et al., 1985). In the rat yolk sac
tumor, the serglycin core protein is translated as a 19-kDa prepro-core
protein, and subsequently processed to a 10-kDa core protein (Bourdon et al., 1985). These results presumably indicate that the
NH
-terminal portion of mouse CTL serglycin is also removed
during maturation similar to rat serglycin.
Glycosaminoglycans on gp600 synthesized by a mouse CTL line, CTLL2, were predominantly chondroitin 4-sulfate, consistent with the previous observation that glycosaminoglycans synthesized by T cells are mainly chondroitin 4-sulfate (Kolset and Gallagher, 1990). Degradation of the chondroitin sulfate moiety resulted in the loss of reactivity with CD44, suggesting that CD44 binds to chondroitin 4-sulfate on gp600/serglycin. It is unlikely that CD44 recognizes the serglycin core protein itself, since CD44 binding to gp600 was completely eliminated by chondroitinase treatment but resistant to protease (data not shown). It appears that CD44 also binds to chondroitin 4,6-sulfate on serglycin, since the soluble CD44 fusion protein binds to secretory granules of mouse bone marrow mast cells where chondroitin 4,6-sulfate type serglycin is known to accumulate (Razin et al. 1982; Stevens et al. 1985). However, binding of CD44 to purified chondroitin 4-sulfate or other chondroitin sulfates was not observed in the present study. In addition, the binding competition assay using fluoresceinated hyaluronate indicated that the affinity, if any, of purified chondroitin sulfates for CD44 is very low when compared to gp600 or hyaluronate. This observation is in agreement with reports by Miyake et al.(1990) and Murakami et al.(1990) but incompatible with three previous reports (Underhill et al., 1983; Aruffo et al., 1990; Sy et al., 1991). Miyake et al.(1990) and Murakami et al. (1990) reported that CD44 binds to hyaluronate but not to chondroitin sulfates. In contrast, it has been reported that CD44 binds to conventional chondroitin sulfates (Underhill et al., 1983; Aruffo et al., 1990; Sy et al., 1991). While it is uncertain whether these discrepancies are due to differences in the CD44 isoforms examined or the cell types or chondroitin sulfates used, we are currently inclined to think that CD44 may recognize a particular conformation of chondroitin 4-sulfate which might be formed by covalent bonding to the serglycin core protein. It may be that certain clusters of chondroitin sulfate chains on the gp600/serglycin core protein are important for high affinity binding of CD44. It is of note that CD44 has been reported to bind to chondroitin sulfate on the class II invariant chain (Naujokas et al. 1993). Although only a single chondroitin sulfate side chain associates with the core protein of class II invariant chain (Sant et al. 1985a, 1985b; Miller et al. 1988), it has been speculated that the lateral association of class II molecules may form clusters of chondroitin sulfate side chains (Brown et al. 1993).
Cell adhesion experiments suggest that
the active form of CD44 can bind gp600/serglycin. The active form of
CD44 is observed on the surface of cytotoxic splenic T cells and
capable of binding ligands during an in vivo allogeneic
response (Lesley et al., 1994), while in vitro stimulated splenocytes is a rich source of gp600/serglycin and
secrete it extracellularly. ()The CD3-dependent granzyme A
release by CTL clones of which CD44 had been activated by IRAWB14
antibody was demonstrated to be significantly augmented by the addition
of serglycin. While it remains to be determined whether
IRAWB14-activated CD44 and physiologically activated CD44 transduce the
same intracellular signal, our results suggest that gp600/serglycin is
involved in CTL activation, and that the binding of gp600/serglycin can
induce a stimulatory signal in T cells. This is consistent with
previous observations that anti-CD44 antibodies can induce cytolytic
activity, granzyme release of CTL clones, and transduction of a
co-stimulatory signal in T cells (Seth et al., 1991;
Galandrini et al., 1993; Shimizu et al., 1989). It is
notable in this regard that only gp600/serglycin enhanced granzyme
release and that hyaluronate had no effect. This may indicate that
gp600/serglycin acts as a principal ligand for CD44 expressed on
cytotoxic T cells.
The results obtained in antibody inhibition and flow cytometry analyses indicate that the gp600 binding domain is located close to the hyaluronate binding domain. However, the binding domains of these two ligands may be different. Alternatively, gp600/serglycin may have a higher affinity for CD44 than hyaluronate, although the binding affinities of gp600 and hyaluronate for CD44 have not been properly determined. Further study is required to understand how CD44 allows differential binding of diverse ligands in various adhesion events.
Due to its acidic nature, serglycin is implicated in packaging and stabilizing basically charged proteases, cytolytic proteins such as perforin, or cytokines within the secretory granules, and also in transporting them outside cells (Masson et al. 1990; Stevens et al. 1988a; Levitt and Olmstead 1986). Since serglycin can bind to cell surface CD44, it may prevent these molecules from random diffusion in the extracellular milieu when they are exocytosed, and help them to target and concentrate on the surface of CD44 positive cells, which would allow efficient delivery and presentation of effector molecules to target cells.
Since CD44 has been thought to play an important role in lymphocyte-high endothelial cell interaction (Jalkanen et al. 1987), it is of interest to investigate whether serglycin is also localized on the surface of endothelial cells. While immunohistological studies using antibody against serglycin have not yet been carried out, the observation that lymphocyte binding to the high endothelial venule is not affected by the anti-CD44 antibody KM201 (Culty et al. 1990), which blocks the binding of CD44 to serglycin, suggests that serglycin may not be directly involved in the recognition of the high endothelial venule by resting lymphocytes. However, in the case of inflamed tissues where the accumulation of serglycin-bearing leukocytes takes place, it is conceivable that serglycin secreted from cells would accumulate around endothelial cells or on the cell surface, thus allowing blood-borne CD44-positive cells to be recruited in situ. Detailed histopathological analysis will be required to resolve this issue.
Although CD44 is implicated in various types of immune responses, not only as an adhesion molecule but also as a signal transducer (Shimizu et al. 1989; Denning et al., 1990; Lesley et al., 1993a), the ligand(s) for CD44 that plays a major role in these responses remains to be elucidated. The ubiquitous expression of known ligands such as hyaluronate, collagen, and fibronectin is apparently incompatible with the cell-type specific function of CD44. In contrast, serglycin is a cell lineage-specific proteoglycan expressed in lymphoid and myeloid cells (Stevens et al. 1988a), and, as shown in our study, secreted extracellularly to bind to cell surface CD44. Such characteristics make serglycin an interesting candidate as a principal ligand for CD44 in immune responses, although the final conclusion awaits experimental verification.
In conclusion, we demonstrate that chondroitin 4-sulfate type serglycin is a novel ligand for CD44. Although the results obtained in the present study indicate that CD44-gp600/serglycin interaction is involved in leukocyte adherence and CTL activation, different cells produce different CD44 isoforms and different types of serglycins, and the significance of the CD44-serglycin interaction may vary in different cell types. The in vivo significance of the CD44-serglycin interaction merits further investigation.